Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations

1-1-1998 Effects of amitraz on insulin and glucagon secretion Ehab Ehab A. H. Iowa State University

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This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Effects of amitraz on insulin and glucagon secretion

by

Ehab A.H. Abu Basha

A thesis submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Major: Physiology (Pharmacology)

Major Professor: Walter H. Hsu

Iowa State University

Ames, Iowa

1998

Copyright © Ehab A.H. Abu Basah, 1998. All right reserved ii

Graduate College Iowa State University

This is to certify that the Master's thesis of

Ehab A.H. Abu Bahsa has met the thesis requirements of Iowa State University

Signatures have been redacted for privacy iii

DEDICATION

I would like to dedicate this thesis to my loving sister, Rania, and to her husband,

Hasan, who were killed in a car accident in February, 1997. They were strong believers and very close friends of mine. They left a lovely daughter, Hala, who lives with my mother and father. I really cannot forget them and they are in my heart and their pictures are always in my mind. Also I would like to dedicate this thesis to my brother, Ghazi, who died in October, 1997. I hope they will have a better life after the judgment day. Finally, I dedicate this thesis, to my father, my mother, and the rest of my sisters and brothers. They all have encouraged and supported me to continue my graduate studies. iv

TABLE OF CONTENTS

ABSTRACT vi

REVIEW OF LITERATURE 1 Physical and chemical characteristics of amitraz 1 Metabolism of amitraz 2 Mechanism of antiparasitic action of amitraz 4 Octopamine in invertebrates and mammals 4 Synthesis and degradation of octopamine 6 Metabolism of octopamine 6 Function of octopamine 8 Octopamine receptors 9 Mechanism of action of octopamine 10 Toxic effects of amitraz and mechanisms of action 10 Characterization of a-adrenergic receptors 13 Classification of ARs 13 a 1-ARs heterogeneity 14 a2-ARs heterogeneity 14 Signal transduction of a1-ARs 17 Signal transduction of a2-ARs 18 Role of a2-adrenergic receptors on endocrine pancreas 19 Effect on insulin secretion 19 Effect on glucagon secretion 21 Effect of amitraz on glucose metabolism and endocrine secretion 22

EFFECTS OF AMITRAZ ON INSULIN AND GLUCAGON 24 SECRETION Introduction 24 Materials and Methods 26 Experimental design 27 Drugs 28 Data expression and statistical analysis 28 Results 29 Effects of amitraz on insulin secretion 29 Effects of amitraz on glucagon secretion 29 Effects of BTS 27271 on insulin secretion 35 Effects of BTS 27271 on glucagon secretion 42 Effects of a-adrenergic receptor antagonists on amitraz-induced 42 inhibition of insulin secretion v

Discussion 51 Conclusion 56

LITERATURE CITED 57

ACKNOWLEDGEMENTS 79 vi

ABSTRACT

The purpose of this study is to investigate the direct effect of amitraz, a formamidine insecticide/acaricide, and its active metabolite BTS 27271 on insulin and glucagon secretions from the perfused rat pancreas. Amitraz and BTS 27271

(10-8, 10-7, 10-6, or 10-5 M) inhibited insulin secretion in a concentration-dependent manner. Amitraz increased glucagon secretion at 10-5 M, whereas BTS 27271 increased glucagon secretion at 1o- 6 and 1o -5 M. Amitraz and BTS 27271-induced decreases in insulin secretion and increases in glucagon secretion were not abolished during the 10-min washout period. Both amitraz and BTS 27271 (10-7,

10-6, or 10-5 M) inhibited arginine-induced insulin secretion and enhanced arginine­ induced glucagon secretion. ldazoxan, an a2N2o-AR antagonist, prevented the inhibitory effect of amitraz on insulin secretion in a concentration-dependent manner, but prazosin, an a 1- and a 2812c-AR antagonist, failed to antagonize the effect of amitraz. These results suggested that: 1) amitraz and BTS 27271 inhibit insulin and stimulate glucagon secretion from the perfused rat pancreas, 2) amitraz inhibits insulin secretion by activation of a 2N 2o-ARs, and 3) amitraz and BTS 27271 may have a high binding affinity to a2-ARs of the pancreatic islets which may be responsible for these effects. 1

REVIEW OF LITERATURE

Amitraz (N'-[2,4-dimethylphenyl]-N-[(2,4-dimethylphenyl)imino]-N­ methylmethanimidamide) is an insecticide/acaricide that belongs to the formamidine family.

Amitraz is widely used to treat mites and ticks in dogs (Mitra et al., 1993;

Dass, 1996), cattle (Curtis, 1985; Mitra et al., 1993; Morrow et al., 1993; Kagaruki,

1996), pigs (Johansson et al., 1980; Curtis, 1985), camels (Jacquiet, 1994), sheep

(Mitra et al., 1993), and goats (Wright et al. , 1988). Amitraz is one of the most widely used therapies for canine demodicosis (Davis, 1985; Duclos et al., 1994;

Medleau and Willemes, 1995; Ristic et al., 1995; Mojzisova et al., 1997).

Demodicosis in goats (Fleisher et al., 1996), ferrets (Noli et al.; 1996), golden

hamsters (Hasegawa, 1995), and cats (Crown and Campbell, 1988) is also treated with amitraz. In addition, amitraz is used to kill arthropods in agricultural and

horticultural plants (e.g., pear paylla, whitefly on cotton, tetanychid and eriophyid

mites on fruits, citrus, and ornamental, and eggs and neonate larvae of cotton

bollworm and tobacco budworm) (Farm Chemicals Handbook, 1996).

Physical and chemical characteristics of amitraz

Amitraz is a highly lipid-soluble compound; thus, it is rapidly absorbed through the cutaneous route (Hsu and Schaffer, 1988; Dobozy, 1990; Aydin et al., 1997), which makes it potentially hazardous to veterinarians, animal caretakers, and pet

owners. Amitraz residue may be present in fruits and crops when amitraz is used as 2 a pesticide in agronomy and horticulture; thus, it poses a potential hazard to the consumers. Amitraz is not stable under acidic conditions.

Metabolism of amitraz

Amitraz (Fig .1, I) is degraded into N'-[2,4-dimethylphenyl]-N­

methylformamidine (BTS 27271 , Fig .1, II), and 2,4-dimethylformanilide (Fig.1, Ill).

BTS 27271 is degraded into 2,4-dimethylformanilide and the latter is further degraded into 2,4-dimethylaniline (Fig .1, IV), and 4-formamido-3-methylbenzoic acid

(Fig.1, V). Finally, derivatives IV and V are converted to 4-amino-3-methylbenzoic acid (Fig.1, VI). All of these compounds are found in tissues (kidney, intestine, lung ,

blood, heart, spleen, and brain) in low amounts (not more than 25 ppb), and are concentrated in the liver 96 h after amitraz administration (Knowles and Benezet,

1981 ).

Amitraz is rapidly absorbed, distributed, metabolized, and eliminated primarily via urine when administered orally (5 mg/kg) to rats . Urinary elimination was 77.6% for amitraz and 88.7% for BTS 27271 , while only 8.9% of amitraz and 4.1% of BTS

27271 were eliminated via feces by 96 h post-treatment (Knowles and Benezet,

1981). Oral administration of amitraz (100 mg/kg) to dogs yields a peak of plasma

concentration after 5 hand a half-life of 24 h (Hugnet et al. , 1996).

Amitraz is degraded into BTS 27271 when given IV (1 mg/kg) to ponies and

sheep. The rate of amitraz metabolism is different in the two species; it is

undetectable in plasma 5 min following administration in sheep, while it is still

detectable for 90 min in ponies. Differences between sheep and ponies in 3

fis~N =CH-N-CH =N ~Cf-h c~ b~ Hs~ I

Fig. 1. Metabolic pathway of amitraz in rats. I) Amitraz, II) N'-[2,4-dimethylphenyl]-

N-methylformamidine (BTS 27271 ), Ill) 2,4-dimethylformanilide, IV) 2,4- dimethylaniline, V) 4-formamido-3-methylbenzoic acid, and VI) 4-amino-3-

methylbenzoic acid (from Knowles and Benezet, 1981). 4 in pharmacokinetics were observed when BTS 27271 was given IV (0.68 mg/kg), with sheep having a larger volume of distribution and faster body clearance than ponies (Pass and Mogg, 1995). Labeled amitraz ([1 4C] amitraz) in Boophilus microplus larvae (cattle tick) is degraded into BTS 27271 and 2,4- dimethylformanilide in different ratios (Fig. 1, II and Ill). 2,4-dimethylformanilide and

2,4-dimethylaniline are found with other unidentified polar and non-polar metabolites.

From identified compounds, only amitraz and BTS 27271 are toxic to the larvae

(Schuntner and Thompson, 1978). BTS 27271 was more potent than amitraz with

regard to miticidal activity (Schuntner and Thompson, 1978) and mammalian toxicity

(Pass and Mogg, 1991; Chen and Hsu, 1994).

Mechanism of antiparasitic action of amitraz

The mechanisms by which formamidines kill arthropods are not completely

understood, however the formamidines mimic the action of octopamine and have a

high affinity for octopamine receptors (Evans, 1980; Singh et al., 1981 ;

Hashemzadeh et al. , 1985; Roder, 1995).

Octopamine in invertebrates and mammals

The biogenic amine octopamine (p-hydroxyethanolamine) (Fig . 2) was first

discovered in the posterior salivary glands of the octopus (Erspamer and Boretti,

1951 ). Octopamine was found in high concentrations in the nervous system of all

invertebrates examined (Axelrod and Saavedra, 1977; Orchard, 1982; Evans, 1985;

Saavedra, 1989). Unlike mammals, little or no catecholamines (epinephrine and

norepinephrine) are present in invertebrates (Evans, 1985). Very low concentrations

of octopamine have been found in 5

fiNl-l:z H~c"'-cooH bV TYROSINE t DD f(Nl-ii

H()C TYRAMINE

t DBH ~~NH

Hv OCTOPAMINE PNMT

OH NH ~~ "'-c~ Hv SYNEPHRINE

Fig. 2. Synthesis pathway of octopamine. The reaction is catalyzed by the following enzymes: DD, DOPA decarboxylase or aromatic amino acid decarboxylase; DBH, dopamine (tyramine) P-hydroxylase; and PNMT, Phenylethanolamine-N-methyl- transferase (from Evans, 1985). 6 the peripheral tissues and brain of mammals (Molinoff et al., 1969; Molinoff and

Axelrod, 1972; Saaverda and Axerlod, 1973). However, in mammals the turn over of octopamine is very rapid (Brandau and Axelord, 1972; Saavedra, 1989).

Synthesis and degradation of octopamine

In the nervous system of vertebrates, octopamine is synthesized from tyrosine and/or tyramine (Brandau and Axelord, 1972; Saavedra and Axelrod, 1976;

Axelord and Saavedra, 1977). Decarboxylation of tyrosine by DOPA decarboxylase

(DD) (Fig. 2) leads to the formation of tyramine. Octopamine is formed through p­

hydroxylation of tyramine (Fig. 2), and by an unusual route via dehydroxylation of epinephrine and norepinphrine (Brandau and Axelord, 1972). The same metabolic

pathway for octopamine was found in the nervous systems of several invertebrates

such as the locust, moth, and fruitfly (Evans, 1985).

Metabolism of octopamine

Octopamine is metabolized by several routes including: 1) oxidative

deamination of octopamine by monoamine oxidase (MAO) to form p­

hydroxymandelic acid (Fig. 3). Oxidative deamination is the most common route for

octopamine in mammals and most invertebrates (Molinoff et al., 1969). Injection of

a MAO inhibitor causes a marked elevation of octopamine in rat and mouse brain

and peripheral tissues (Brandau and Axelord, 1972; Harmer and Horn, 1976). 2) N­

acetylation of octopamine by N-acetyltransferase to form N-acetyloctopamine (Fig.

3) is a major metabolism route in some invertebrates such as insects (Evans, 1985).

This route was also demonstrated in mammalian tissues (Wu and Boulton, 1979;

Yang and Neff, 1976). 3) sulfate and p-alanine conjugation (Fig. 3) are an important 7

OH

H~~~:ROXYMANDELIC ACID

/ o:OHc

Fig. 3. The possible routes of metabolism of octopamine. 1) monoamine oxidase activity, 2) N-acetyltransferase activity, 3) sulphate conjugation, and 4) p-alanine conjugation (from Evans, 1985). 8 route of octopamine metabolism in the lobster (Kennedy, 1977; Kennedy, 1978). 4)

N-methylation of octopamine by phenylethanolamine-n-methyl-transferase (PNMT)

(Fig. 2) to synephrine (N-methyl octopamine) and ~-hydroxylation of octopamine to norepinephrine is another minor route of octopamine metabolism (Saavedra, 1989;

Axelrod and Saavedra, 1977).

Function of octopamine

It has been well established that octopamine functions as a neurotransmitter, a neuromodulator, and a neurohormone in the invertebrate (Axelrod and Saavedra,

1977; Orchard, 1982; Evans, 1985; Bicker and Randolf, 1989; Saavedra, 1989).

Octopamine is a neurotransmitter in the firefly lantern and controls the time of the light flashes (Carlson, 1969) and it also controls the release of hyperlipemic hormone (increase the level of lipid) in the hemolymph during the flight from

Corpoora Cardiaca (Orchard, 1982; Evans, 1985). Modulation of the myogenic rhythm and potentiation of neuromuscular transmission are examples of the neuromodulator function of octopamine (Orchard, 1982; Evans, 1985). Octopamine is released during the initial period of flight and under stress conditions in insect hemolymph, suggesting a role of octopamine as a neurohormone (Evans, 1985).

The physiological role of octopamine in mammals is not clear (Harmar and

Horn, 1976; Saavedra, 1989). However, octopamine is present in high concentration in mammals experiencing pathological conditions such as phenylketonuria, hepatic pathology, depression, aggression, stress, migraine, epilepsy, Parkinson's disease, and hypertension, suggesting that octopamine may play a pathophysiological role in these conditions (Axelrod and Saavedra, 1977; Saavedra, 1989). 9

Octopamine receptors

Octopamine receptors have been studied in both neural (Nathanson and

Greengard, 1973; Evans and O'shea, 1978; Roder and Grweecke, 1990; Roder,

1995) and non-neural tissues in invertebrates (Evans, 1981; Evans, 1985). There are three classes of octopamine receptors in the locust extensor-tibiae neuromuscular (Evans, 1981). Octopamine receptors are classified as 1) octopamine receptor 1 (OAR1), 2) presynaptic octopamine receptor 2A (OAR2A), and

3) postsynaptic octopamine receptor 28 (OAR2s).

OAR1 can be distinguished from OAR2 on the basis of the affinity for different agonists and antagonists. Chlorpromazine and yohimbine are better antagonists than metoclopramide for OAR1, while the opposite is true for OAR2. Also clonidine is a much better agonist than naphazoline for OAR1, while the converse is true for

OAR2 (Evans, 1981; Evans, 1985). In similar pattern, OAR2A can be distinguished from OAR28. Cyproheptadine, mianserin, and metoclopramide are better antagonists for OAR2A than OAR2s. On the other hand, chlorpromazine is a better antagonist for OAR2B. Naphazoline is a better agonist than tolazoline for OAR2A, while the converse is true for OAR2s (Evans, 1981; Evans, 1985).

Octopamine receptors have similar pharmacological features to a-adrenergic receptors (ARs) in vertebrates (Evans, 1981; Evans, 1985), since they can be blocked by a-AR antagonists such as phentolamine but not by p-AR antagonists such as propranolol (Evans, 1985). Clonidine and tolazoline (a-AR agonists) have agonistic activity on octopamine receptors while isoproterenol (a P- AR agonist) does not have this activity (Evans, 1985). Octopamine receptors can be distinguished 10 from ARs in terms of amine specificity. Octopamine receptors prefer mono-phenolic amines with a single hydroxyl group on the aromatic ring, while ARs prefer two hydroxyls on the ring (Evans, 1985).

Mechanism of action of octopamine

Octopamine mediates it's effect through activation of adenylate cyclase

(Evans, 1985) via stimulation of a special octopamine-sensitive adenylate cyclase

(Nathanson, 1979; Evans, 1980; Saavedra, 1989) in both neural and non-neural tissues, which results in an increase in the formation of cyclic AMP. The octopamine-sensitive adenylate cyclase in the invertebrate has been demonstrated to be the site of action for pesticides (Nathanson, 1979; Saavedra, 1989).

Octopamine receptors are thought to be an ideal target for specific insecticides such as the formamidine pesticides: chlordimeform (COM), N-dimethylchlordimeform

(DCDM), and amitraz (Evans, 1985; Roder, 1995).

Toxic effects of amitraz and mechanisms of action

Earlier studies suggested that toxic effects of formamidines in mammals include: persistent activation of monoamines by inhibiting monoamine oxidase (Aziz and Knoweles, 1973; Benzent and Knowles, 1976; Matsumura and Beeman, 1976); inhibition of oxidative phosphorylation (Abo-Khatwa and Hollingworth, 1974); anticholinergic activity (Wang et al. , 1975); local anesthesia (Lund et al., 1978;

Gilbert and Mack, 1989); and antihistaminic activity (Pass and Seawright, 1982;

Pass and Mogg, 1991).

Over sixteen cases of amitraz toxicity in humans have been reported (FDA

Veterinarian, 1989; Ros and Aken, 1994; Kennel et al. , 1996; Jorens et al. ; 1997; 11

Aydin et al., 1997). The clinical signs include hyperglycemia, bradycardia, hypotension, miosis, headache, nausea, drowsiness, vomiting, polyuria, rash, stiffness, swelling, and coma. The toxic effects of amitraz in animals include hyperglycemia, central nervous system (CNS) depression, bradycardia, hypertension, polyuria, mydriasis, vomiting, delayed gastrointestinal transit, bloat, and colic (Dobozy, 1990).

While most of the toxic signs resulting from exposure to amitraz are similar between humans and animals, hypotension was reported in humans, while both hypertension (Hsu et al., 1986; Cullen and Reynoldson, 1987; Cullen and

Reynoldson, 1988) and hypotension (Pascoe and Reynoldson, 1986; Reynoldson and Cullen, 1996) were reported in animals. The hypertensive effect of amitraz may be attributed to activation of a-ARs in the vascular bed, while the hypotensive and bradycardic effects could be attributed to a2-AR-mediated decreases in sympathetic outflow from the CNS and increases in vagal tone (Clark and Hall, 1969; Schmitt et al., 1970; Antonaccio et al. , 1973). The decreased sympathetic outflow is believed to originate from both pre- and postsynaptic a2-ARs in the pontomedullary region of the CNS (McCall et al., 1983; Van Zwieten and Timmermans, 1983). Activation of the presynaptic a2-ARs located on the adrenergic terminals also inhibits peripheral adrenergic neurotransmission (Langer, 1980; Starke, 1980; De Jonge et al. , 1980).

The increase of vagal tone by a2-agonists may be due to a facilitation of the

baroreceptor reflex (Hucht et al. , 1981; Kitagawa and Walland, 1982). Miosis,

reported in humans could be attributed to a decrease in the sympathetic outflow, 12 while mydriasis, reported in animals could be attributed to a decrease in the parasympathetic outflow.

The toxic effects of amitraz resemble those induced by the a2-AR agonists, xylazine and clonidine (Hsu and Kakuk, 1984). In addition, the a2-AR antagonists such as yohimbine, but not the a1-AR antagonists, attenuate or block several of the effects of amitraz such as: CNS depression (Hsu and Hopper, 1986; Schaffer et al.,

1990), bradycardia (Hsu and Kakuk, 1984; Hsu and McNeel, 1986; Hsu et al., 1986;

Schaffer et al., 1990; Cullen and Reynoldson, 1987), hypertension (Hsu et al., 1986;

Cullen and Reynoldson, 1987), mydriasis (Hsu and Kakuk, 1984), delayed gastrointestinal transit (Hsu and Lu, 1984; Hsu and McNeel, 1985; Robert and

Argenzio, 1986; Hsu et al., 1987 ), and glucose intolerance (Smith et al., 1990).

Death in rats caused by intraperitoneal injections of amitraz is reduced by yohimbine (Moser and McPhail, 1985). Amitraz causes a dose-dependent inhibition of [3H]clonidine and [3H]yohimbine binding to a2-AR in mouse brain (Costa et al.,

1989a) and rat brain (Costa et al., 1989b). Amitraz inhibits epinephrine-induced canine platelet aggregation in a dose-dependent manner, and also inhibits

[3H]yohimbine binding to a2-ARs (Koeiman and Hsu, 1991 ). These findings suggest that the a2-ARs mediate a number of the toxic effects of amitraz and that a2-AR antagonists have the ability to block these effects.

Amitraz has been shown to decrease hepatic glutathione concentration by activating hepatic a2-ARs similar to epinephrine and clonidine (Costa et al., 1991 ).

Neither amitraz nor its metabolites are mutagens (Tudek et al., 1988). 13

Characterization of a-adrenergic receptors

Classification of ARs

The ARs were initially classified as a- and P-ARs subtypes, based on their pharmacological response to five catecholamines on eight different physiological functions (Ahlquist, 1948). Subsequently, the p-ARs were divided into p1 and p2 subtypes based on their affinity for agonists (Lands et al., 1967), and selective antagonists and by binding studies (Minneman et al., 1979). Later, a third P-AR was isolated and designated as p3-AR (Emorine et al, 1989). a-ARs were first classified into a 1 and a2 subtypes (Delbarre and Schmitt, 1973). Based on their anatomical location, a-ARs were subclassified as presynaptic (prejunctional) a-ARs and postsynaptic (postjuctional) a-ARs (Dubocovich and Langer, 1974). Langer (1974) suggested that the postsynaptic a-ARs be referred to a1, while the presynaptic a­

ARs be referred to a 2. Subsequently, a-ARs were classified according to their physiological functions namely that a 1-ARs mediate excitatory effects, while a 2-ARs mediate inhibitory effects (Berthelsen and Pettinger, 1977).

Both prazosin and yohimbine antagonize norepinephrine-induced presser responses (Drew and Whiting, 1979) indicating that both a1-ARs and a 2-ARs mediate postsynaptic excitatory responses. Therefore, the a-ARs subclassification based on anatomical location or physiological function does not appear to be sufficient. Pharmacological classification in terms of the affinities of agonists and antagonists was proposed as a better method by which to subclassify a-ARs

(Starke, 1981; Ruffolo et al., 1991). The a1-ARs are activated by methoxamine, 14 cirazoline, and phenylephrine and antagonized by prazosin and WB 4101. The a2-

ARs are activated by clonidine, xylazine, medetomidine and UK 14304 and antagonized by yohimbine, rauwolsin, and idazoxan. a1-ARs heterogeneity

Based on pharmacological characteristics and molecular cloning the a1-ARs have been further classified into three subtypes: a 1A-, a 18-, a 10-ARs (Table 1). The a1A-ARs are characterized by a selective agonistic activity for oxymetazoline and cirazoline (Horie et al., 1995), and a selective antagonistic activity for 5- methylurapidil and (+)-Niguldipine (Gross et al., 1988; Boer et al., 1989). The gene for a 1A-ARs has been cloned from the bovine brain complementary DNA (Schwinn et al., 1990).

The a 18-ARs have no selective agonists or antagonists (Table 1). However, a 18-ARs have a low affinity for WB 4101 , 5-methylurapidil, and phentolamine

(Cotecchia et al., 1988; Lomasney et al., 1991). The gene for a 18-ARs has been cloned from hamster complementary DNA (Cotecchia et al., 1988).

A third distinct subtype designated as a 10-AR was cloned from rat brain complementary DNA (Lomasney et al., 1991; Perez et al., 1991). This receptor has no selective agonists or antagonists. arARs heterogeneity

Based on pharmacological characteristics and molecular cloning a2-ARs have been subclassified into four subtypes: a2A-, a2s-, a2c-, a20-ARs (Bylund, 1992; 15

Table 1: Subtypes of a-Adrenergic Receptors

Gene Located a-ARs Selective Selective Tissue in human subtype agonists antagonists localization chromosome # a1A 8 Oxymetazoline 5- Heart, liver, , cirazoline Methylurapidil, cerebellum, (+)-Niguldipine cerebral cortex, prostate, lung , vas deferens

a10 5 None WB 4101 (low Kidney, affinity) spleen, aorta, lung, cerebral cortex a10 20 None None Aorta, cerebral cortex, prostate, hippocampus

a2A * 10 Oxymetazoline BRL 44408 Platelet, cerebral cortex, locus cervleus, spinal cord

a2B 2 None Prazosin, Liver, kidney ARC-239, BRL 42992

a2c 4 None Prazosin, Cerebral ARC- 239, cortex Rauwolsine, WB 4101 , BAM 1303

* a2A and aw are species orthologs, e.g. human and pigs have a2A whereas rats and cattle have aw. The aw receptors have no selective agonists or antagonists. 16

Bylund et al., 1994). The a2A-ARs, are characterized by a high affinity for oxymetazoline and BRL 44408 (Table 1) and low affinity for prazosin and ARC-239, are expressed in human platelets and HT29 (a human adenocarcinoma cell line)

(Bylund et al., 1988). The gene for the a2A-ARs has been cloned from human

(KobilKa et al., 1987), pig (Guyer et al., 1990), and guinea pig (Svensson et al. ,

1996).

The a2s-ARs are found in neonatal rat lung and the NG 108 cell line

(neurobalstoma x glioma hybrid cell line) (Bylund et al. , 1988). The gene has been cloned from human (Lomasney et al., 1990; Weinshank et al. , 1990), rat (Zeng et al.,

1990), mouse (Chruscinski et al. , 1992) and guinea pig (Sevnsson et al., 1996). The a28-ARs have no selective agonists (Table 1), but they do have a relatively high affinity for prazosin, BRL 42992, and ARC-239, and low affinity for oxymetazoline

(Bylund et al., 1988).

The third a2-AR subtype, a2c-AR, has been identified from an opossum kidney-derived cell line (OK cells) (Murphy and Bylund, 1988). This receptor subtype has no selective agonists (Table 1) , and has similar characteristics to a 28-

AR (e.g. high affinity of prazosin), but the affinity is intermediate between a2A- and a2s-ARs (Murphy and Bylund, 1988). a2c-ARs are distinguished from a28-ARs by their higher affinity for the antagonists rauwolscine, WB 4101 , and BAM 1303

(Blaxall et al., 1991). The gene for a2c-ARs has been cloned from human (Regan et al. , 1988), rat (Lanier et al. , 1991 ), mouse (Link et al. , 1992), and guinea pig

(Svensson et al. , 1996). 17

The a20-ARs have been found in bovine pineal gland (Simonneaux et al.,

1991), rat submaxillary glands (Michel et al., 1989), and the p-cell tumor-derived cell line (RINm5F) (Remaury and Paris, 1992). SK&F 104078, an a2-antagonist, blocks a2A-,a2s-, a2c-ARs, but not a20-AR (Bylund and Iversen, 1990). a 20-ARs have no selective agonists or antagonists, and the gene has been cloned from the rat (Lanier et al., 1991) and mouse (Link et al., 1992). a2A- and a 20-ARs are species orthologs

(O'rourke et al , 1994), which means that a species may have either a2A or a 20 , but not both. Humans and pigs have a2A-ARs, whereas rats and cattle have a20-ARs

(O'rourke et al , 1994).

Signal transduction of a1-ARs

The a 1-ARs are coupled to a variety of regulatory systems (Table 2). These include:

1) The mobilization of intracellular Ca2+ from the endoplasmic reticulum. The activation of phospholipase C (PLC) through the Gq family leads to hydrolysis of membrane-bound polyphosphoinositides, which results in the generation of diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3) (Lefowitz et al., 1996;

Piascik et al. , 1996). IP3 stimulates Ca2+ release from the endoplasmic reticulum, whereas DAG activates protein kinase C (PKC) that can phosphorylate many regulatory proteins (Berridge, 1993).

2) The stimulation of phospholipase A2 (PLA2), which leads to the release of free arachidonate to produce prostaglandins and leukotrienes via cyclooxygenase and lipoxygenase pathways, respectively. 18

3) The stimulation of phospholipase D (PLO), that leads to hydrolysis of phosphatidylcholine and yields phosphatidic acid (PA). PA acts as a second messenger by releasing Ca2+ from the endoplasmic reticulum. It is also metabolized to DAG (Lefowitz et al., 1996).

Signal transduction of a2-ARs

The a2-ARs couple to a variety of effectors (Table 2) . Activation of a2-ARs leads to:

1) The inhibition of adenylate cyclase activity and the reduction of cyclic AMP formation in different target cells including human platelets (Mills, 1975), neuroblastoma x glioma hybrid cells (Sabol and Nirenberg, 1979), rat renal cortex

(Woodcock and Johnston, 1982), porcine thyroid (Muraki et al, 1984 ), and rat pancreatic islets (Yamazaki et al, 1982).

2) The activation of G-protein-gated K+ channels which leads to membrane hyperpolarization (Egan et al. , 1983; Morita and North, 1981; Lefowitz et al.,1996).

3) The inhibition of voltage-gated Ca2+ Channels (Horn and McAffee, 1980; Holz et al., 1986; Lefowitz et al.,1996).

4) Other messenger systems linked to the a2-ARs: a) The acceleration of Na+/H+ exchange (Sweatt et al., 1985), resulting in an increase in intracellular pH, and Na+ concentration. b) The stimulation of PLA2 (Sweatt et al., 1986). c) The stimulation of PLO (MacNulty et al. 1990). d) The stimulation of PLC (Cotecchia et al. , 1990). 19

The activation of these pathways by the a2-ARs is pertussis-toxin sensitive,

suggesting that GTP-binding proteins of the G/G0 subfamily are responsible for

eliciting these effects (Limbird, 1988; Piascik et al., 1996). All four subtypes of a2-

ARs have no difference or selectivity in signal transduction mechanisms (Bylund et

al., 1994).

Role of aradrenergic receptors on the endocrine pancreas

Effect on insulin secretion

The endocrine pancreas is innervated by the sympathetic nervous system

(Smith and Porte, 1976), which plays an important role in the regulation of the secretion of pancreatic hormones through the release of catecholamines.

Stimulation of splanchnic nerves (Porte et al., 1973), and the ventromedial hypothalamus (Forhman and Bernard is, 1971) leads to activation of the sympathetic nervous system, thereby decreasing insulin secretion (Porte et al., 1966).

The a2-ARs are responsible for the inhibition of glucose-stimulated insulin secretion (Nakaki et al., 1983; Ismail et al., 1983; Skoglund et al., 1986; Schuit and

Pipeleers, 1986; Hirose et al, 1997). Nonselective a-AR agonists such as epinephrine and norepinphrine, and selective a2-AR agonists such as clonidine, xylazine, medetomidine, oxymetazoline, and UK 14.304 inhibit insulin secretion

(Ismail et al., 1983; Angel et al., 1988; Niddam et al., 1990; Angel et al., 1990; Chen and Hsu, 1994). The a2-AR antagonists but not the a 1-AR antagonists counteract the inhibitory effect on insulin secretion. The a2-AR agonists cause temporary diabetes mellitus in cattle (Hsu and Hummel, 1981), whereas a2-AR antagonists 20 improve insulin secretion and glucose disposal in type II diabetic patients (Kashiwagi et al., 1986; Ortiz-Alonso et al., 1991).

It is well established that the a2-ARs mediate the inhibition of insulin secretion from p-cells of the pancreas. One possible mechanism for this effect is through the inhibition of adenylate cyclase, an action mediated by a PTX-sensitive G-protein

(Chen and Hsu, 1994; Horise et al, 1997). However, it is still not known which a2-AR subtypes mediate this inhibition. Only two published studies have dealt with the characterization of the a2-AR subtype that mediates the inhibition of insulin secretion

(Angel et al, 1990; Hirose et al, 1993a). These authors suggested that a2KAR is responsible for the inhibition of insulin secretion. However, in both studies oxymetazoline was used as an a2-AR agonist, despite the fact that oxymetazoline has potent a 1-AR agonistic activity (Garicia et al., 1995). In the first study a specific a2-AR antagonist was not used since WB 4101 antagonizes both a2A- and a2c-ARs

(Hieble and Ruffolo, 1995). In the second study idazoxan was used as a selective a2A-antagonist and yohimbine as a nonselective antagonist, while it is known that idazoxan antagonizes aw- more than a2A-ARs, and yohimbine antagonizes a2A­ more than aw-ARs (Satoh and Takayanagi, 1992). In addition, rodents have aw­

ARs, but not a2A-ARs since a2A and aw are species orthologs (O'Rourke et al,

1994). Therefore, it is likely that aw-ARs are the receptors that mediate the inhibition of insulin in rodents. 21

Effect on glucagon secretion

The a 2-AR agonists such as oxymetazoline and clonidine stimulate glucagon secretion from a-cells of the pancreatic islet in the rat. The a2-AR antagonists but not the a 1-AR antagonists counteract the stimulatory effect on glucagon secretion

(Gotch et al. , 1988; Horise et al. , 1993a; 1993b; 1993c).

It is possible that the effect of the az-AR agonists on glucagon secretion is through a direct stimulation of the pancreatic a-cells or indirectly through an inhibition of insulin secretion, since it is known that insulin inhibits glucagon secretion

(Waeber et al., 1997). Several studies suggested that a2-AR agonist-induced glucagon secretion is mediated mainly through the direct stimulation of a-cells

(Tominaga et al., 1987; Hirose et al , 1993c). Glucagon secretion was induced by az­

AR agonists in the absence of any changes in insulin secretion (Hirose et al, 1993c).

However, this study did not demonstrate the inhibitory effect of az-AR agonists on

insulin secretion due to the very low basal insulin secretion rate. The very low

insulin concentration might be due to an insensitive insulin assay that could not detect changes in low basal concentrations of insulin. In another study, increases in glucagon secretion induced by norepinephrine were still present after the destruction

of pancreatic p-cells by streptozocin in rats (Tominaga et al., 1987; Hirose et al;

1993c). However, norepinephrine was used rather than a selective az-AR agonist

and p-antatgonist was not used to exclude the p-adrenergic effect. It is likely that

glucagon secretion is mediated mainly through the direct stimulation of a-cells and to

a lesser extent through the indirect inhibition of insulin secretion. 22

Effect of amitraz on glucose metabolism and endocrine secretion

The effects of amitraz on glucose metabolism and endocrine secretion were first noted in dogs through the clinical use of this pesticide. Topical administration of amitraz to dogs at concentrations of~ 227 ppm of an aqueous suspension caused hyperglycemia (Hsu and Schaffer, 1988; and Dobozy, 1990) and hypoinsulinemia

(Hsu and Schaffer, 1988). Normal dogs dipped in an aqueous suspension of 555 ppm (twice the recommended dose) showed no increase in insulin release following

IV injection of glucose (0.6 g/kg) that was given 4 h after amitraz application (Hsu and Schaffer, 1988). It is not known how much cutaneous absorption of amitraz takes place through dipping; however, because amitraz is highly lipid soluble, a significant amount of amitraz is expected to be absorbed into the body.

Oral administration of amitraz (100 mg/kg) increases plasma glucose concentration and decreases insulin secretion in dogs, which were antagonized by atipamezole, an a 2-AR antagonist (50µg/kg, IM) (Hugnet et al., 1996). Intravenous administration of amitraz into rats at dosages of 0.1, 0.3, and 1 mg/kg followed by IV injection of glucose (1 g/kg) caused a dose-dependent inhibition of plasma insulin and glucose intolerance (Smith et al., 1990). Intravenous administration of amitraz at 1 mg/kg completely suppressed the insulin secretion induced by IV glucose

(Smith et al., 1990). In addition, amitraz and its active metabolite, BTS 27271,

inhibited 3-isobutyl-1-methyl xanthine (IBMX) (a phosphdiesterase inhibitor)­ stimulated insulin secretion from a ~-cell line, RINm5F (Chen and Hsu, 1994).

Amitraz, BTS 27271, and medetomidine, an a2-AR agonist, inhibited IBMX-induced

increases in intracellular cyclic AMP concentrations (Chen and Hsu, 1994). In 23 pertussis toxin (PTX)-pretreated cells, neither amitraz nor BTS 27271 attenuated the

IBMX-induced increase in insulin secretion (Chen and Hsu, 1994). These results suggest that amitraz and its active metabolite BTS 27271 induce hyperglycemia through the inhibition of insulin secretion, which is mediated by a2-ARs. One possible mechanism for this effect is through the inhibition of adenylate cyclase, an action mediated by PTX-sensitive G-proteins (Chen and Hsu, 1994).

Since hyperglycemia is found in human cases of amitraz poisoning, the exposure to this pesticide would be expected to pose a great hazard to patients with type II diabetes, who already have impaired insulin secretion and/or utilization.

There are at least 14 million people with type II diabetes in the U.S.A. Exposure of these individuals to amitraz and its active metabolite might reduce their insulin secretion and further hamper glucose metabolism. The a2-AR agonists cause a greater suppression of insulin secretion from human patients with type II diabetes than from normal subjects (Okada et al., 1986; and Koev et al., 1989). 24

EFFECTS OF AMITRAZ ON INSULIN AND GLUCAGON

SECRETION

Introduction

Amitraz (N'-[2,4-dimethylphenyl]-N-[(2,4-dimethylphenyl)imino]-N­ methylmethanimidamide) is a formamidne insecticide/acaricide used to treat mange in animals (Farmer and Seawright, 1980) and kill arthropods in agronomy and horticulture practice (Farm Chemicals Handbook, 1996). The insecticide/acaricide actions of amitraz may be mediated by octopamine receptors (Evans, 1980; Singh et al., 1981, Hashemzadeh et al., 1985; Roder, 1995).

Amitraz is rapidly absorbed, distributed, metabolized, and eliminated primarily via urine when administered orally to mammals. The degradation products present in the urine include N'-[2,4-dimethylphenyl]-N-methylformamidine (BTS 27271 ), 2,4- dimethylformanilide, 2,4-dimethylformanilide, 2,4-dimethylaniline, 4-formamido-3- methylbenzoic acid, 4-amino-3-methylbenzoic acid, and several unknowns (Knowles and Benezet, 1981 ). BTS 27271, one of the metabolites of amitraz, has been found to be more potent than amitraz with regard to its miticidal activity (Schuntner and

Thompson, 1978) and mammalian toxicity (Pass and Mogg, 1991; Chen and Hsu,

1994).

Over sixteen cases of amitraz toxicity in humans have been reported (FDA

Veterinarian, 1989; Ros and Aken, 1994; Kennel et al., 1996; Jorens et al.; 1997; and Aydin et al., 1997). Symptoms include hyperglycemia, bradycardia, hypotension, 25 miosis, headache, nausea, drowsiness, vomiting, polyuria, rash, stiffness, swelling, and coma. The toxic effects of amitraz in animals include hyperglycemia, central nervous system (CNS) depression, bradycardia, hypertension, polyuria, mydriasis, vomiting, delayed gastrointestinal transit, bloat, and colic (Dobozy, 1990). The toxic effects of amitraz resemble those induced by the a2-AR agonists, xylazine and clonidine (Hsu and Kakuk, 1984). In addition, a2-AR antagonists such as yohimbine, but not the a1-AR antagonists, attenuate or block several of the effects of amitraz such as: CNS depression (Hsu and Hopper, 1986; Schaffer et al., 1990), bradycardia (Hsu and Kakuk, 1984; Hsu and McNeel, 1986; Hsu et al., 1986;

Schaffer et al., 1990; Reynoldson and Cullen, 1987), hypertension (Hsu et al., 1986;

Reynoldson and Cullen, 1987), mydriasis (Hsu and Kakuk, 1984), delayed gastrointestinal transit (Hsu and Lu , 1984; Hsu and McNeel, 1985; Robert and

Argenzio, 1986; Hsu et al., 1987 ), and glucose intolerance (Smith et al., 1990).

Amitraz causes a dose-dependent inhibition of [3H]clonidine and [3H]yohimbine binding to a2-AR mouse brain (Costa et al. , 1990) and rat brain(Costa et al. , 1988).

These findings suggest that the a2-ARs mediate a number of the effects of amitraz and that the a2-AR antagonists have the ability to block these effects.

Amitraz causes hyperglycemia and hypoinsulinemia when used topically (Hsu and Schaffer, 1988), orally (Hug net et al. , 1996), and intravenously (Smith et al. ,

1990). These effects of amitraz are similar to those induced by the a2-AR agonists.

In addition, yohimbine, an a2-AR antagonist, antagonized the effect of amitraz on insulin secretion, whereas prazosin, an a 1-AR antagonist, did not (Smith et al. , 1990; 26

Chen and Hsu, 1994). Amitraz and its active metabolite, BTS 27271, inhibits intracellular cyclic AMP concentration and insulin secretion from a p-cell line,

RINm5F, (Chen and Hsu, 1994). Pretreatment of RINm5F cells with pertussis toxin

(PTX) antagonized the effect of amitraz and BTS 27271 on insulin secretion. These results suggest that amitraz and its active metabolite BTS 27271 induce hyperglycemia through inhibition of insulin secretion, which is mediated by a 2-ARs.

One possible mechanism for this effect is through the inhibition of adenylate cyclase, an action mediated by PTX-sensitive G-proteins (Chen and Hsu, 1994).

The a2-AR agonists such as oxymetazoline and clonidine stimulate glucagon secretion from a-cells of the pancreatic islet in the rat. The a2-AR antagonists but not the a1-AR antagonists counteract the stimulatory effect on glucagon secretion

(Gotoh et al., 1988; Hirose et al., 1993a; 1993b; 1993c).

Since previous studies have not investigated the direct effect of amitraz and its active metabolite BTS 27271 on the secretion of the insulin and glucagon from the pancreas, the present study was designed to address this question and in addition, to characterize the a-ARs that mediate the effect of amitraz on insulin secretion from the pancreas.

Materials and Methods

Male Sprague-Dawley rats (300-400 g) were used. The rats were

anesthetized with sodium pentobarbital (60 mg/kg, ip), maintained at 37C 0 on a hot

plate, and were kept alive during the experiment. Pancreatic perfusion was

performed as previously described (Yang and Hsu, 1995). After cannulation of the 27 celiac artery, the rat pancreas was immediately perfused with Krebs-Ringer

Bicarbonate (KRB) solution supplemented with 20 mM N-2-hydroxyethylpiperazine­

N'-2-ethanesulfonic acid, 5.5 mM glucose, and 0.2% bovine serum albumin as a basal medium. The KRB was maintained at pH 7.4 and continuously aerated with

95% 02 - 5% C02.

Experimental design

The first 15-20 min of perfusion was considered as an equilibration period.

Subsequently, the effluent fluid was collected every min from the cannula in the portal vein. The flow was maintained at 1 ml/min. In experiment 1, after a baseline period of 10-min, the medium containing amitraz (10-8 , 10-7, 10-6, or 10-5 M) was administered for 10-min, and was followed by a washout period during which the basal medium was administered for 10-min. In experiment 2, after a baseline period of 10-min, the medium containing BTS 27271 (10-9, 10-8, 10-7, 10-6, or 10-5 M), the active metabolite of amitraz, was administered for 10 min, and was followed by a washout period during which the basal medium was administered for 10-min. In

experiment 3, the pancreas was pretreated for 10-min with a medium containing either the a-AR antagonist idazoxan (10-8, 10-7, or 10-6 M), or prazosin (10-6 M). This was followed by the medium containing amitraz (10-6 M) and idazoxan or prazosin for 10-min, and the basal medium for another 10-min for the washout period. In all

experiments, 1 mM arginine was administrated at the end of experiment for 10-min

to stimulate both insulin and glucagon secretion, in order to confirm that the tissue

retained the normal secretory capacity under the experimental conditions. The 28 effluent fluids were kept at 4°C and subsequently assayed within 6 h for insulin and glucagon using radioimmunoassy (RIA).

Drugs

The following drugs were used: amitraz (Agr Evo, Pikeville, NC), BTS 27271

HCI (Nor-Am Chemical, Wilmington, DE) , idazoxan (Reckitt & Colman

Pharmaceutical, England), and prazosin HCI (Pfizer, Groton, CT). All drugs were dissolved in distilled H20 except amitraz, which was dissolved in dimethyl sulfoxide

(DMSO) to make stock solution of 5x10-3 M. These solutions were further diluted with the KRB (basal medium) to attain the appropriate concentrations. Control experiments were performed following the same protocol except that KRB was used in place of the drugs.

Data expression and statistical analysis

The effluent concentrations of insulin and glucagon were expressed as a percentage of the baseline level (mean of 10 baseline values) in means± SE. The area under the curve (treatment period area, washout period area, and arginine period area) was calculated using a scanning program (Sigma Scan; Jandel, Corte

Madera, CA). Data were analyzed using the SAS Proc General Linear Means

(GLM) procedure. The factors were drugs (2 levels), dose (5 levels), and area (3

levels). The general design is a split-plot factorial design with repeated measures.

Individual mean comparisons were calculated using the F test. The significance

level was set at P < 0.05. 29

Results

Effects of amitraz on insulin secretion

Insulin secretion remained constant during the first 30-min in the control group receiving 5.5 mM glucose only. Administration of 1 mM arginine in the control group increased insulin secretion by - 200% of the baseline level at the end of the experiment (Fig. 4). Amitraz caused a concentration-dependent inhibition of insulin secretion. Amitraz (10-8 , 10-7 , 10-6 , or 10-5 M) significantly inhibited insulin secretion, and reached a maximal inhibition of 33%, 47%, 60%, and 77% of the baseline level, respectively (Figs. 4 and 5). The effluent concentrations of insulin remained low during the 10-min washout period in all the groups, although it was expected that the effluent concentrations of insulin would return to baseline level. Amitraz (~ 10-7 M) significantly inhibited arginine-induced insulin secretion (Figs. 4 and 5).

By calculating the area under the curve for the amitraz treatment and by using the corresponding area of the control groups as 100%, amitraz (10-8, 10-7, 1o- 6, or

10-5 M) significantly inhibited insulin secretion in a concentration-dependent manner

(Fig. 6). Also by calculating the area under the curve for the arginine treatment and by using the corresponding area of the control groups as 100%, amitraz (10-7, 10-6, or 10-5 M) significantly inhibited arginine-induced insulin secretion (Fig. 7).

Effects of amitraz on glucagon secretion

The effluent concentrations of glucagon remained constant in the control group receiving 5.5 mM glucose only. Administration of 1 mM arginine into the control group increased glucagon secretion with a peak of - 470% of the baseline level (Fig. 8). Amitraz (10-8, 10-7, or 10-6 M) did not cause any significant increases 30

260

240 .-.. Amitraz Arginine 1 mM 0~ 220 Q Q ~ 200 II -+-Control -cu 180 ti) ---A 10-aM cu 160 ._.m -.-A 10·7 M c 140 0 ·- 120 .....Cl) (.) Cl) 100 tn ·-c 80 -:::::s 60 ti) c - 40 0~ 20

0 0 5 10 15 20 25 30 35 40 Time (min)

Fig. 4. The effects of 10-8 M and 10-7 M amitraz (A) on insulin secretion from the perfused rat pancreas. After a baseline period of 10 min, amitraz (shown in the heavy line) was given for 10 min, followed by 10 min of the washout period. At the end of the experiment, 1 mM arginine (shown in the heavy line) was given for 10 min. Values are shown as mean± SE (n=4). 31

260

240 Amitraz Arginine 1 mM 220 -0~ 0 0 200 ~ II -ca 180 -+--Control tnca ---A 10-&M m 160 ._.. -.-A 10-5 M c 140 0 ·-....Q) 120 ...u Q) 100 UJ ·-c 80 -:::::s tn 60 c - 40 0~ 20

0 0 5 10 15 20 25 30 35 40 Time (min)

Fig. 5. The effects of 10-6 M and 10-5 M amitraz (A) on insulin secretion from the perfused rat pancreas. After a baseline period of 10 min, amitraz (shown in the heavy line) was given for 10 min, followed by 10 min of the washout period. At the end of the experiment, 1 mM arginine (shown in the heavy line) was given for 10 min. Values are shown as mean ± SE (n=5). 32

120

-~0 100 0 0 ""'"II * -0 ~ 80 ....c: 0 * (.)._. c: * 0 60 ·-....Q) ~ (.) * Q) 40 "'c: ·--::s c: -"' 20 ~0

0 ~- A 10-s M A 10·7 M A 10-e M A 10·5 M

Fig. 6. A summary of amitraz-induced inhibition of insulin secretion. Values are shown as mean ±SE (n=4-5). *P<0.05, compared with the control group. 33

120 ..., s::: CJ) ...,E ns 100 CJ) L. I- CJ) - s::: ~ ·- 0 s::: 0 80 ·- 0 C>"""" <1!. C>s::: ...,~ ·-L. 0s::: 60 :::J (.) * "'C CJ) s::: s::: O·- * ·-..., ·-s::: ~ C> 40 (.) L. * enCJ)~ ·-s::: -:::J 20 ti) s:::

0~ 0 A 10-s M A 10·7 M A 10-6 M A 10·5 M

Fig. 7. A summary of 1 mM arginine-induced insulin secretion in groups that were exposed to amitraz previously. Values are shown as mean± SE (n=4-5). *P<0.05, compared with the control group. 34

2000

'O' C»' 0 .....0 II 1500 __._ A 10·7 M -ca tn e,ca --- A 10-8 M c --+- Control ·-.....0 ! 1000 (,) Amitraz Arginine 1 mM Cl) c "'0 CJ) ca (,) 500 ::;, (!)- '?ft.

0 0 5 10 15 20 25 30 35 40 Time (min)

Fig. 8. The effects of 10-8 M and 10-7 M amitraz (A) on glucagon secretion from the perfused rat pancreas. After a baseline period of 10 min, amitraz (shown in the heavy line) was given for 10 min, followed by 10 min of the washout period. At the end of the experiment, 1 mM arginine (shown in the heavy line) was given for 10 min. Values are shown as mean ± SE (n=4 ). 35

In glucagon secretion during the treatment and the washout periods (Figs. 8, and 9).

However, Amitraz (10-5 M) significantly increased glucagon secretion with a peak of

265% of the baseline level (Fig. 9). The effluent concentrations of glucagon level remained high during the 10-min washout period (Fig. 9). Amitraz (~ 10-7 M) significantly enhanced arginine-induced glucagon secretion (Figs. 8 and 9).

By calculating the area under the curve for the arginine treatment and by using the corresponding area of the control groups as 100%, Amitraz (10-7, 10-6, or

10-5 M) significantly enhanced arginine-induced glucagon secretion in a concentration-dependent manner (Fig. 10).

Effects of BTS 27271 on insulin secretion

In a pattern similar to amitraz, BTS 27271, the active metabolite of amitraz, caused a concentration-dependent inhibition of insulin secretion. The BTS 27271

(10-9 M) did not cause any significant inhibition of insulin secretion (Fig. 11 ).

However, BTS 27271 administrated at 10-8, 10-7, 10-6, or 10-5 M significantly inhibited insulin secretion, with a maximal inhibition of 34%, 52%, 72%, and 85% of the baseline level, respectively (Figs. 12 and 13). The effluent concentrations of insulin remained low in all groups during the 10-min washout period. The BTS 27271 (~ 10-

7 M) caused a significant inhibition of arginine-induced insulin secretion (Figs. 12 and

13).

By calculating the area under the curve for the BTS 27271 treatment and by using the corresponding area of the control groups as 100%, BTS 27271 (10-8, 10-7 ,

10-6 , or 10-5 M) significantly inhibited insulin secretion in a concentration-dependent manner (Fig. 14). Also by calculating the area under the curve for the arginine 36

2000 Amitraz Arginine 1 mM

-0~ 0 0 T- II 1500 -ca tn -.-A 10·5 M ca ._..m ---A 10-& M c 0 -+-Control ·-..... 1000 ! CJ Cl) tn c 0 C> ca 500 CJ :::::s C>-

0~

0 0 5 10 15 20 25 30 35 40 Time (min)

Fig. 9. The effects of 10-6 M and 10-5 M amitraz (A) on glucagon secretion from the perfused rat pancreas. After a baseline period of 10 min, amitraz (shown in the heavy line) was given for 10 min, followed by 10 min of the washout period. At the end of the experiment, 1 mM arginine (shown in the heavy line) was given for 10 min. Values are shown as mean± SE (n=5). 37

...., 600 c: (1) ....,E ns ....(1) 500 * I- (1) c: - ·-c: ~0 ·-oe>o 400 <( ii C>- c: e ·-....,.... c: :::J 0 300 "'C (.) c: (1) .2...., c: (1) ·-c: ....(.) ·-C> 200 (1) .... (/) ~ c: 0 C> ns 100 (.) :::J -(!)

0~ 0 A 10-s M A 10·7 M A 10-6 M A 10·5 M

Fig. 10. A summary of 1 mM arginine-induced glucagon secretion in groups that were exposed to amitraz previously. Values are shown as mean± SE (n=4-5).

*P<0.05, compared with the control group. 38

260

240 BTS Arginine 1 mM

-0~ 220 0 0 T"" 200 _._control II -cu 180 9 ti) ---BTS 10- M mcu 160 -c 140 0 ·-... 120 ...G) (J 100 enG) ·-c 80 -::::s 60 ti) c - 40 0~ 20

0 0 5 10 15 20 25 30 35 40

Time (min)

Fig. 11. The effect of 10-9 M BTS 27271 on insulin secretion from the perfused rat pancreas. After baseline period of 10 min, BTS (shown in the heavy line) was given for 10 min, followed by 10 min of the washout period. At the end of experiment, 1 mM arginine was given for 10 min (shown in the heavy line). Values are shown as mean± SE (n=3-4). 39

260

240 Arginine 1 mM .-.. BTS 0~ 220 0 0 200 ~ II -ca 180 -+- Control U) ca 160 -e- BTS 10-8 M ._..m _.__ BTS 10-7 M c 140 0 ·-.... 120 eu Cl> 100 Ul ·-c 80 -:::::s 60 U) c - 40 0~ 20

0 0 5 10 15 20 25 30 35 40 Time (min)

Fig. 12. The effects of 10-8 Mand 10-7 M BTS 27271 on insulin secretion from the perfused rat pancreas. After baseline period of 10 min, BTS (shown in the heavy line) was given for 10 min, followed by 10 min of the washout period. At the end of experiment, 1 mM arginine was given for 10 min (shown in the heavy line). Values are shown as mean ± SE (n=3-4 ). 40

260

240 BTS Arginine 1 mM .-.. 220 0~ 0 0 200 ~ II -ca 180 --+- Control ti) ca 160 ---- BTS 10-8 M .._.m __...._ BTS 10-7 M c 140 0 ·-... 120 Cl) '- CJ Cl) 100 tn c 80 ·-::::s ti) 60 c - 40 0~ 20

0 0 5 10 15 20 25 30 35 40 Time (min}

Fig. 13. The effects of 10-6 Mand 10-5 M BTS 27271 on insulin secretion from the perfused rat pancreas. After baseline period of 10 min, BTS (shown in the heavy line) was given for 10 min, followed by 10 min of the washout period. At the end of experiment, 1 mM arginine was given for 10 min (shown in the heavy line). Values are shown as mean ± SE (n=3-5). 41

120

-0~ 0 0 100 ~ -II ...,0... c: 80 * 0 (.) * -c: ·-...,0 60 ...Cl) (.) Cl) * "'c: 40 ·--:::s * c: -"' 20 ~0

0 BTS 10-a M BTS 10-7 M BTS 10-6 M BTS 10-s M

Fig. 14. A summary of BTS 27271-induced inhibition of insulin secretion. Values are shown a mean ± SE (n=3-5). *P<0.05, compared with the control group. 42 treatment and by using the corresponding area of the control groups as 100%,

BTS 27271 (10-7, 10-6, or 10-5 M) significantly inhibited arginine-induced insulin secretion (Fig. 15).

Effects of BTS 27271 on glucagon secretion

BTS 27271 (10-9, 10-8, or 10-7 M) did not cause significant increases in glucagon secretion during the treatment and the washout periods (Figs. 16 and 17).

However, BTS 27271 (10-7 M) enhanced arginine-induced glucagon secretion (Fig.

16). BTS 27271 (10-6 and 10-5 M) significantly increased glucagon secretion with a peak of 275% and 228% of the baseline level, respectively. The effluent concentrations of glucagon remained high during the 10-min washout period in both groups (Fig. 18). Both concentrations of BTS 27271 significantly enhanced arginine­ induced glucagon secretion (Fig. 18).

By calculating the area under the curve for the BTS 27271 treatment and by using the corresponding area of the control groups as 100%, BTS 27271(10-7,10-6 , or 10-5 M) significantly enhanced arginine-induced glucagon secretion in a concentration-dependent manner (Fig. 17).

Effects of a-adrenergic receptor antagonists on amitraz-induced inhibition of insulin secretion

Amitraz (10-5 M) alone inhibited insulin secretion by 45 ± 7% of baseline level

(Fig. 6). Pretreatment of the pancreas with an a 2-AR antagonist idazoxan (10-8 M) for 10 min, did not significantly changed amitraz -induced inhibition of insulin secretion (Fig. 20), whereas idazoxan (10-7 and 10-5 M) antagonized the effect of amitraz on insulin secretion in a concentration-dependent manner (Figs. 20 and 22). 43

120 ...c: Q) E ...ca 100 Q).... I- Q) - c: ~ ·- 0 c: 0 80 ·-C> "'(-'0 .._ II <- C>c: ...e ·- c: 60 ....:s 0(.) * "'C Q) c: c: O·- ·- c: ...Q) ·-C> 40 ...... (.) < CJ)Q) - ·-c: * -:s,,, 20 * c:

~0 0 BTS 10-s M BTS 10·7 M BTS 10-6 M BTS 10-s M

Fig. 15. A summary of arginine (1 mM)-induced insulin secretion in groups that were exposed to BTS 27271 previously. Values are shown as mean± SE (n=3-5).

*P<0.05, compared with the control group. 44

2000

-0~ 0 0 ~ II 1500 -cu U) ---- BTS 10-9 M mcu -+-Control -c 0 ·-..Cl) 1000 ~ CJ BTS Arginine 1 mM Cl) U) c 0 C) cu 500 CJ ::::s (!)-

0~

0 0 5 10 15 20 25 30 35 40 Time (min)

Fig. 16. The effect of 10-9 M BTS 27271 on glucagon secretion from the perfused rat pancreas. After a baseline period of 10 min, BTS (shown in the heavy line) was given for 10 min, followed by 10 min of the washout period. At the end of the experiment, 1 mM arginine (shown in the heavy line) was given for 10 min. Values are shown as mean ± SE (n=3-4 ). 45

2000 .-.. ';ft 0 0 T-9 ~ BTS 10·7 M .!!. 1500 ca ---BTS 10-s M ca -+-Control ._..m"' c 0 +;e 1000 (.) BTS Arginine 1 mM Cl) c "'0 g> 500 (.) :l C>- ';ft

0 5 10 15 20 25 30 35 40 Time (min)

Fig. 17. The effects of 10-8 Mand 10-7 M BTS 27271 on glucagon secretion from the perfused rat pancreas. After a baseline period of 10 min, BTS (shown in the heavy line) was given for 10 min, followed by 10 min of the washout period. At the end of the experiment, 1 mM arginine (shown in the heavy line) was given for 10 min. Values are shown as mean± SE (n=3-4). 46

2000

-0~ 0 0 "'l""' II BTS Arginine 1 mM -ca 1500 ti) ca --.- BTS 10-5 M .._..m c -e- BTS 10-6 M 0 ..... -+- Control ·-Cl) 1000 u~ Cl) "'c 0 C>ca 500 u :l C)-

0~ 0 0 5 10 15 20 25 30 35 40 Time (min)

Fig. 18. The effects of 10-6 Mand 10-5 M BTS 27271 on glucagon secretion from the perfused rat pancreas. After a baseline period of 10 min, BTS (shown in the heavy line) was given for 10 min, followed by 10 min of the washout period. At the end of the experiment, 1 mM arginine (shown in the heavy line) was given for 10 min. Values are shown as mean± SE (n=3-4). 47

600 ...... c C1) ...... E ns 500 C1) L. * I- C1) c- ·-Co~ ·-eo0 400 <( ii en- * c e ·-L...... c :::J 0 300 ""C (.) c C1) .2 c * ...... C1) ·-c L.u ·-en 200 C1) L. (/) ~ c 0 en ns (.) 100 :::J -(!)

~0 0 BTS 10-a M BTS 10·7 M BTS 10-& M BTS 10-5 M

Fig. 19. A summary of 1 mM arginine-induced glucagon secretion in groups that were exposed to BTS 27271 previously. Values are shown as mean± SE (n=3-5).

*P<0.05, compared with the control group. 48

260

240 Arginine 1 m M .-.. 220 0~ 0 ldazoxan 0 200 "'"'"II -ca 180 -+- Ida. 10-6 M & A 10-6 M ti) ca Ida. 10-7 M & A 10-6 M m 160 ._.. __.__ Ida. 10-7 M & A 10-6 M c 140 0 -a- A 10-6 M ~ ·-Cl) 120 ~ (.) Cl) 100 tn c 80 ·--::s ti) 60 c - 40 0~ 20

0 0 5 10 15 20 25 30 35 40 Time (min)

Fig. 20. The effects of ldazoxan (lda.)(10-8, 10-7, 10-6 M) (shown in the heavy line) on amitraz (A)(1 o-6 M)-induced insulin secretion from the perfused rat pancreas.

After a pretreatment period of 10 min with idazoxan, amitraz (shown in the heavy line) was given for 10 min, followed by 10 min of the washout period. At the end of the experiment, 1 mM arginine (shown in the heavy line) was given for 10 min.

Values are shown as mean± SE (n=4). 49

260

240 Amitraz 10-6 M Arginine 1 mM 220 -0~ 0 0 200 "t""9 II -ca 180 ti) --+- Control ca 160 -...-- Pra. 10-6 M ._.m - A 10-6 M c 140 ·-0 .....cu 120 (.) cu 100 U) c 80 ·--::::s ti) 60 c - 40 0~ 20

0 0 5 10 15 20 25 30 35 40 Time (min} Fig. 21. The effects of prazosin (Pra.) (10-6 M) (shown in the heavy line) on amitraz

(10-6 M)-induced inhibition of insulin secretion from the perfused rat pancreas. After a pretreatment period of 10 min with prazosin, amitraz (shown in the heavy line) was given for 10 min, followed by 10 min of the washout period. At the end of the experiment, 1 mM arginine (shown in the heavy line) was given for 10 min. Values are shown as mean± SE (n=4). 50

120

-. • Treatment Period 0~ 0 100 0 T- bdJ Washout Period II -0 ...... 80 c 0 .._..0 * c 60 ·-.....0 * ...Cl) (.) Cl) en 40 c ·--:J ti) -c 20 0~

0 A 10..e M Pra. 10..e M Ida. 10-a M Ida. 10-7 M Ida. 10..e M + + + + A 10..e M A 10..e M A 10..e M A 10..e M

Fig. 22. A summary of the effect of a.-adrenergic receptor antagonist (ldazoxan

(Ida.), or Prazosin (Pra.)) on 1 µM amitraz (A)-induced insulin secretion during the treatment and washout periods. Values are shown as mean± SE (n=4-5). *P<0.05, compared with the control group. 51 ldazoxan (10-7 M) abolished or attenuated this effect during the treatment and the washout periods, respectively (Figs. 20 and 22). ldazoxan (10-6 M) abolished the effect of amitraz during both the treatment and the washout periods (Figs. 20 and

22). Prazosin (10-6 M), an a 1-AR antagonist, failed to antagonize the effect of amitraz (Fig . 21). By calculating the area under the curve for the arginine treatment and by using the corresponding area of the control groups as 100%, idazoxan (10-7 and 10-6 M) abolished the inhibitory effect of amitraz (10-6 M) on 1 mM arginine­ induced insulin secretion (Fig. 23).

Discussion

This is the first study to demonstrate the direct effect of amitraz and BTS

27271, the active metabolite of amitraz, on insulin and glucagon secretion from the pancreas. These results are consistent with the findings of previous in vivo (Hsu and Kakuk, 1984; Hsu et al., 1986; Schaffer et al., 1990; Reynoldson and Cullen,

1997) and clonal ~-cell (Chen and Su, 1994) studies with respect to the effects of amitraz and BTS 27271 on insulin secretion.

Amitraz and BTS 27271 (10-8 - 10-5 M) caused a concentration-dependent inhibition of insulin secretion, and the effect of BTS 27721 was slightly more potent than that of amitraz. These results are consistent with those of another study that demonstrated the inhibitory effect of amitraz and BTS 27271 on insulin secretion from a rat p-cell line RINm5F (Chen and Hsu , 1994). The other metabolites of amitraz were not examined in the present study, as it is known that BTS 27271 is the only metabolite of amitraz that inhibits insulin secretion from RINm5F cells (Chen and Hsu, 1994). BTS 27271 has been found to be more potent than amitraz with 52

120 ...c: Q) E ...ns 100 Q) ....a.. Q) - c: ~ ·- 0 c: 0 80 ·- 0 C>"t""' a.. II c: ...e ·-a.. c:0 60 :J (.) "C c: Q) o·-c: ·- c: ...Q) ·-C> 40 a.. a.. (.) <( enQ) - ·-c: -:J 20 ti) c:

~0 0 A 101 M Pra. 101 M Ida. 10..a M Ida. 10-7 M Ida. 101 M + + A101 M A 101 M

Fig. 23. A summary of 1 mM arginine-induced insulin secretion in groups that were previously exposed to a-adrenoceptor antagonist (ldazoxan (Ida.) or Prazosin (Pra.)) and amitraz (A). Values are shown as mean ±SE (n=4-5). *P<0.05, compared with the control group. 53 regard to miticidal activity (Schuntner and Thompson, 1987) and mammalian toxicity

(Pass and Mogg, 1991 ).

Amitraz and BTS 27271 increased glucagon secretion and enhanced arginine-induced glucagon secretion. These results were not unexpected since other studies showed that a2-AR agonists such as oxymetazoline and clonidine stimulate glucagon secretion (Gotoh et al., 1988; Hirose et al, 1992; 1993a; 1993b).

However, it is clear from these results that the effect of amitraz and BTS 27271 on insulin secretion is more potent than that on glucagon secretion.

Amitraz is a highly-lipid soluble compound; which is rapidly absorbed through cutaneous tissues (Hsu and Schaffer, 1988; Dobozy, 1990; Aydin et al., 1997), and thus, it is potentially hazardous to veterinarians, animal caretakers, and pet owners.

Over sixteen cases of amitraz toxicity in humans in which hyperglycemia was the most prominent sign have been reported (FDA Veterinarian, 1989; Ros and Aken,

1994; Kennel et al. , 1996; Jorens et al.; 1997; and Aydin et al., 1997). Since hyperglycemia is found in most of the human cases of amitraz poisoning, exposure to this pesticide would be expected to pose a greater hazard to patients with type II diabetes, who already have impaired insulin secretion and/or utilization. a2-AR agonists cause a greater suppression of insulin secretion from human patients with type II diabetes than from normal subjects (Okada et al. , 1986; and Koev et al.,

1989). Exposure of patients with type II diabetes to amitraz and its active metabolite may reduce their insulin secretion and further hamper glucose metabolism.

In both the amitraz and the BTS 27271 treated groups, the inhibition of insulin secretion persisted. The effluent insulin concentrations remained low during the 10- 54 min washout period, although they were expected to return to baseline level after the washout of the pesticide. To answer the question whether this effect is due to insufficient washout time, we prolonged the washout period to 20-min, however, the effluent concentrations remained low and did not return to the baseline level (data not shown). We speculate that this phenomenon may be due to a high binding affinity of amitraz and BTS 27271 to their receptors. The effluent concentrations of glucagon also remained high during the 10-min washout period for both the amitraz and the BTS 27271 treated groups. In vivo administration of amitraz caused a dose­ dependent inhibition of 3H-clonidine to a 2-ARs in rat brain, and the recovery of 3H­ clonidine binding occurred only 72 h following amitraz administration (Costa et al.,

1988). The persistent effect of amitraz and BTS 27271 on the secretion of insulin and glucagon should prompt careful handling of this pesticide to avoid the toxicity.

By calculating the area under the curve for the arginine treatment and by using the corresponding area of the control groups as 100%, amitraz and BTS

27271 (1 o-7, 10-6, or 10-5 M) significantly inhibited arginine-induced insulin secretion.

However, when insulin secretion was expressed as the percentage increase above the pre-arginine value, the groups that had been previously exposed to amitraz and

BTS 27271 were not significantly different from the control groups. All groups had

~ 200% increase over the baseline level in response to 1 mM arginine. Thus, the effect of amitraz and BTS 27271 on arginine-induced insulin secretion depends upon

how the data are expressed. 55

ldazoxan, an a2A120-AR antagonist, blocked the inhibitory effect of amitraz on insulin secretion in a concentration-dependent manner, but prazosin, an a 1- and a 2812c-AR antagonist, failed to antagonize the effect of amitraz. These results suggest that a2A/2o-ARs mediate the inhibitory effects of amitraz on insulin secretion.

Our present findings are consistent with those of the previous reports in which a 2-

ARs mediate the actions of amitraz in animals (Hsu and Kakuk, 1984; Hsu et al.,

1986; Schaffer et al., 1990; Reynoldson and Cullen, 1996) and a p-cell line (Chen and Hsu, 1994). The a2A120-ARs are orthologs, i.e., a species may have either a2A or a20 but not both; rodents have a20-ARs, but not a2A-ARs (O'Rourke et al, 1994).

Thus a 20-ARs should be the receptors that mediate the inhibition of insulin in rodents.

We did not study the effect of idazoxan or prasozin on the amitraz induced­ increase in glucagon secretion, because we used amitraz at a concentration of 10-5

M, which decreased insulin secretion by 45 ± 7% of the baseline level but did not cause significant increases in glucagon secretion. We intended to use amitraz (10-5

M) for the antagonism study, but idazoxan (10-6 M) failed to antagonize the effects of amitraz at this concentration (data not shown).

lmidadozline binding sites on pancreatic p-cells have been reported

(Remaury and Herve, 1992; Chan et al., 1995; Morgan et al., 1995). Many a 2-AR agonists (e.g., oxymetazoline and clonidine), a2-AR antagonists (e.g., efaroxan, DG-

5128, and idazoxan), and the nonselective a-AR antagonist phentolamine are 56 imidazoline compounds. It is well established that imidazoline compounds such as phentolamine (Schulz and Hasselblatt, 1989), efraroxan (Chan and Morgan, 1990), and DG-5128 (Chan et al. , 1991) stimulate insulin secretion. In the present study idazoxan (~10-6 M), failed to stimulate insulin secretion. These results are consistent with those of another study that demonstrated that idazoxan failed to alter insulin secretion or arginine-induced glucagon secretion (Mourtada et al., 1997).

Conclusion

Our findings suggest that amitraz inhibits insulin secretion by the activation of a2N 2o-ARs. Amitraz and BTS 27271 inhibit arginine-induced insulin secretion and enhance arginine-induced glucagon secretion. These effects may result from the high binding affinity of amitraz and BTS 27271 to the a2-ARs of the pancreatic islets.

Since amitraz exerts a2-AR agonistic activities, specific a 2-AR antagonists may be useful in treating amitraz toxicity including hyperglycemia and hypoinsulinemia. The results of several human studies indicate a possible role of the a2-adrenegic receptors in the impaired p-cell function in type II diabetic patients (Robertson et al.,

1976, Kashiwagi et al , 1986, Ortiz-Alonso et al. , 1991). It is likely that these patients are more sensitive to amitraz than normal subjects. Further studies are needed to prove or disprove this hypothesis. 57

LITERATURE CITED

Abo-Khatwa, N. , and Hollingworth, R. M. (1974). Pesticidal chemicals affecting some

energy-linked functions of rat liver mitochondria in vitro. Bull. Environ. Contam.

Toxicol. 12, 446-454.

Ahlquist, R. S. (1948). A study of the adrenotropic receptors. Am. J. Physiol. 153,

583-600.

Angel, I., Bidet, S., and Langer, S. Z. (1988). Pharmacological characterization of

alpha-2 adrenceptor agonists . . J. Pharmacol. Exp. Ther. 246, 1098-1103.

Angel, I., Niddam, R., Langer, S. Z. (1988). Involvement of alpha-2 adrenergic

receptor subtypes in hyperglycemia. J. Pharmacol. Exp. Ther. 254, 877-882.

Antonaccio, M. J. , Robson, R. D., and Kerwin, L. (1973). Evidence for increased

vagal tone and enhancement of baroreceptor activity after xylazine in

anesthetized dogs. Eur. J. Pharmacol. 23, 311-315.

Axelrod, J., and Saavedra, J.M. (1977). Octopamine. Nature. 265, 501-504.

Aydin, K., Kurto·glu, S., Poyrazo·glu, M. H., and Uzum, K. et al. (1997). Amitraz

poisoning in children: clinical and laboratory findings of eight cases. Hum.

Exp.Toxicol. 16, 680-682.

Aziz, S. A, and Knoweles, C. 0. (1973). Inhibition of monoamine oxidase by the

pesticide chlordimeform and related compounds. Nature. 24, 417-418.

Benzent, H. J. , and Knowles, C. 0 . (1976). Inhibition of rat brain monoamine oxidase

by formamidines and related compounds. Neurophamacology. 15, 369-373. 58

Berthelsen, S., and Pettinger, W. A. (1977). A functional basis for the classification

of alpha adrenergic receptors. Life Sci. 21, 595-606.

Berridge, M. J. (1993). Inositol triphosphate and calcium signaling. Nature. 361, 315-

325.

Bicker, G., and Randolf M. (1989). Chemical codes for the control of behavior in

arthropods. Nature. 337, 33-39.

Blaxall,H. S., Murphy, T. J., Baker, J. C., Ray, C., and Bylund, D. B. (1991 ).

Characterization of the alpha-2C adrenergic receptor subtype in the opossum

kidney and in the OK cell line. J. Pharmacol. Exp. Ther. 259, 323-329.

Boer, R., Grasseger, A., Schudt, C.H., and Glossman, H. (1989). (+)-Niguldipine

binds with very high affinity to Ca++ channels and to a subtype of alpha1-

adrenoceptor subtypes. Eur. J. Pharmacol. 172, 131-145. Bylund, D. B. (1992).

Subtypes of alpha-1 and alpha-2 adrenergic receptors. Fed. Am. Soc. Exp. Biol. J.

6, 832-839, 1992.

Brandau, K., and Axelrod, J. (1972). The biosynthesis of octopamine. N. S. Arch.

Pharm. 273, 123-133.

Bylund, D. B., Eikenberg, D. C., Hieble, J. P., Langer, S. Z., Lefkowitz, R. J.,

Minneman, K. P., Molinoff, P. B., Ruffolo, R.R., and Trendelenburg, U. (1994).

International Union of Pharmacology nomenclature of adrenoceptors. Pharmacol.

Rev. 46, 121-136.

Bylund, D. B., and Iversen, L. (1990). Low affinity of SK&F 104078 for bovine pineal

and rat sublingual alpha-20 adrenergic receptors (A2AR): a putative presynaptic

alpha-2 subtype. Pharmacologist. 32, 144. 59

Bylund, D. B., Ray-Prenger, C., and Murphy, T. J. (1988). Alpha-2A and alpha-2B

adrenergic receptors subtypes: Antagonist binding in tissues and cell lines

containing only one subtype. J. Pharmacol. Exp. Thre. 245, 600-607.

Carlson, A. D. (1969). Neural control of firefly luminescence. Adv. Insect Physiol. 6,

51-96.

Chan, S. L., Stillings, M. R., Morgan, N. G. (1991). Mechanisms involved in

stimulation of insulin secretion by the hypoglycemic alpha-adrenergic antagonist,

DG-5128. Biochem. Biophys. Res. Commun. 176, 1545-1551 .

Chan, S. L., and Morgan, N. G. (1990). Stimulation of insulin secretion by efaroxan

may involve interaction with potassium channels. Eur. J. Pharmacol. 176, 97-101.

Chen, T. H., and Hsu, W. H. (1994). Inhibition of insulin release by a formamidine

pesticide amitraz and its metabolites in a rat beta-cell line: an action mediated by

alpha-2 adrenoceptors, a GTP-binding protein and a decrease in cyclic AMP. J.

Pharmacol. Exp. Ther. 271, 1240-1245.

Chruscinski, A. J., Link, R. E. , Daunt, D. A. , Barsh, G. S., and Kobilka, B. K. (1992).

Cloning and expression of the mouse homolog of the human alpha-2C2

adrenergic receptor. Biochem. Biophys. Res. Commun. 186, 1280-1287.

Clark, K. W., and Hall, L. W . (1969). Xylazine-a new sedative for horses and cattle.

Vet. Res. 85, 512-517.Cotecchia, S., Kobilka, B. K., Daniel, K. W., Nolan, R. D.,

Lapetina, E. G., Caron, M. G., Lefkowitz, R. J., and Regan, J. W. (1990). Multiple

second messenger pathways of a 2-adrenergic receptor subtypes expressed in

eukaryotic cells. J. Biol. Chem. 265, 63-69. 60

Costa, L. G. , Olibet, G., Wu, D.S., and Murphy, S. D. (1989a). Acute and chronic

effects of the pesticide amitraz on alpha 2-adrenoceptors in mouse brain. Toxicol.

Lett. 47, 135-143.

Costa, L. G. , Wu, D.S., Olibet, G., Murphy, S. D. (1989b). Formamidine Pesticides

and Alpha2-Adrenoceptors: Studies With Amitraz and Chlordimeform in Rats and

Development of a Radioreceptor Binding Assay. Neurotoxicol teratol 11, 405-411 .

Costa, L. G., Gastel, J., and Murphy, S. D. (1991). The formamidine pesticides

chlordimeform and amitraz decrease hepatic glutathione in mice through an

interaction with alpha 2-adrenoceptors. J. Toxicol. Environ. Health. 33, 349-358.

Cotecchia, S., Schwinn, D. A. , Randall, R. R. , Lefkowitz, R. J., et al., Caron, M. G.,

and Kobilka, B. K. (1988). Molecular cloning and expression of the cDNA for the

hamster a 1-adrenergic receptor. Proc. Natl. Acad. Sci. U.S.A. 85, 7159-7163.

Cowan, L.A., and Campbell, K. (1988). Generalized demodicosis in a cat

responsive to amitraz. J. Am. Vet. Med. Assoc. 192, 1442-1444.

Cullen, L. K., Reynoldson, J. A. (1987). Cardiovascular and respiratory effects of the

acaricide amitraz. J. Vet. Pharmacol. Ther. 10, 134-143.

Cullen, L. K., and Reynoldson, J. A. (1988). Cardiovascular responses to amitraz in

the presence of autonomic antagonists and agonists. Arch. Int. Pharmacodyn.

Ther. 296, 45-56.

Curtis, R. J. (1985). Amitraz in the control of non-ixodide ectoparasites of livestock.

Vet. Parasitol. 18, 251-264.

Dass, S. S. (1996). Effect of a herbal compound for treatment of sarcoptic mange

infestations on dogs. Vet. Parasitol. 63, 303-306. 61

Davis, D. A. (1985). The treatment of canine demodecosis with amitraz. J. S. Afr.

Vet. Assoc. 56, 43-46.

De Jonge, A., and Timmermans, P. B, and Van Zwieten, P.A. (1982). Quantitative

aspects of alpha-adrenergic effects induced by clonidine-like imidazlidines. II.

central and peripheral bradycardia activities. J. Pharmacol. Exp. Ther. 222, 712-

719.

Delbarre, B., and Schmitt, H. (1973). A further attempt to characterize sedative

receptors activated by clonidine in chickens and mice. Eur. J. Pharmcol. 22, 355-

359.

Dobozy VS. Mitaban safety. (1990). DVM. 13, 54-55.

Drew, G. M., and Whiting, S. B. (1979). Evidence for two distinct types of

postsynaptic a-adrenoceptors in vascular smooth muscle in vivo. Br. J.

Pharmacol. 67, 207-215.

Dubocovich, M. L., and Langer, S. Z. (1974). Negative feed-back regulation of

noradrenaline release by nerve stimulation in the perfused cat's spleen:

differences in potency of phenoxybenzamine in blocking the pre- and post­

synaptic adrernergic receptors. J. Physiol. 237, 505-519.

Duclos, D. D., Jeffers, J. G., and Shanely K. J. (1994). Prognosis for treatment of

adult-onset demodicosis in dogs. J. Am. Vet. Med. Assoc. 204, 616-619.

Egan, T. M., Henderson, G., North, R. A. , and Williams, J. T. (1983). Noradrenaline­

mediated synaptic inhibition in rat locus coeruleus neurons. J. Physiol. 345, 477-

488. 62

Emorine, L. J., Marullo, S. , Briend-Sutren, M. M., Patey, G., Tate, k., Delavier­

Klutchko, C., and Strosberg, A. D. (1989). Molecular characterization of the

human beta 3-adrenergic receptor. Science. 245, 1118-1121.

Erspamer, V., and Boretti, G. (1951). Identification and characterization by paper

chromatography of enteramine, octopamine, tyramine, histamine, and allied

substance in extracts of vertebrates and invertebrates. Arch . Int. Pharmacodyn.

88, 296-332.

Evans, P. D., and Gee, J. D. (1980). Action of formamidine pesticides on

octopamine receptors. Nature. 287, 60-62.

Evans, P. D. (1980). Biogenic amines in the insect nervous system. Adv. Insect

Physiol. 15, 317-473.

Evans, P.O., and O'shea, M. (1978). The identification of an octopamine neuron and

the modulation of a myogenic rhythm in the locust. J. Exp. Biol. 73, 235-260.

Evans, P. D. (1981). Multiple receptor types for octopamine in locust. J. Physiol.

Land. 318, 99-122.

Evans, P. D. (1985). Octopamine. In Comprehensive Insect Physiology,

Biochemistry and Pharmacology. (G . A. , Kerkut, and G., Glibert, Eds) pp. 499-

530. Pergamon.Press, Oxford .

Farm Chemicals Handbook (1996), C 23, Meister Publishing Co., Willoughby, OH.

Farmer, H., and seawright, A. A. (1980). The use of amitraz (N-(2,4-dimethylphenyl)­

N-[((2,4-dimethylphenyl)imino)-N-methylmethanimidamide] in demodicosis in

dogs. Aus. Vet. J. 57, 537-541.

FDA Veterinarian. (1989). Adverse Drug Reactions. Vol. IV, NO. 5. 63

Fleischer, P., Lukesova, D., and Skrivanek, M. (1996). [First report of demodicosis in

goats in the Czech Republic]. 41, 289-293.

Forhman, L.A., and Bernardis, L. L. (1971). Effect of hypothalamic stimulation on

plasma glucose, insulin and glucagon levels. Am. J. Physiol. 221, 1596-1603.

Garicia-Sainz, j. A. Olivares-Reyes, J. A., Macias-Sliva, M. Villalobos-Molina, R.

(1995). Characterization of the arn-adrenoceptors of catfish hepatocytres:

functional and binding studies. Gen. Comp. Endocrinol. 97, 111-120.

Gilbert, M. E., and Mack, C. M. (1989). Enhanced susceptibility to kindling by

chlordimeform may be mediated by a local anesthetic action.

Psychopharmacology. 99, 163-167.

Gotoh, M., lguchi, A., and Sakamoto, N. (1988). Central versus peripheral effect of

clonidine on hepatic venous plasma glucose concentrations in fasted rats.

Diabetes. 37, 44-49.

Gross, G., Hanft, G., and Rugevices, C. (1988). 5-methylurapidil discriminates

between subtypes of alpha1-adrenoceptor. Eur. J. Pharmacol. 151, 333-335.

Guyer, C. A., Horstman, D. A., Wilson, A. L., Clark, J. D., Cragoe, E. J., and Limbird,

L. E. (1990). Cloning, sequencing and expression of the gene encoding for the

porcine platelet a 2-adrenergic receptor. J. Biol. Chem. 265, 17307-17317.

Harmar, A. J., and Horn, A. S. (1976). Octopamine in mammalian brain: Rapid post

mortem increases and effects of drugs. J. Neurochem. 26, 987-993.

Hasegawa, T. (1995). A case report of the management of demdicosis in the golden

hamster. J. Vet. Med. Sci. 57, 337-338. 64

Hashemzadeh, H., Hollingworth, R. M., and Voliva, A. (1985). Receptor for 3H­

octopamine in the adult firefly light organ. Life Sci. 37, 433-440.

Hieble, J. P., and Ruffolo, R. R. (1995). a 2-adrenoceptors. In G-Protein Coupled

Transmembrane Signaling Mechanisms. (R. R., Rufflo, and J. P., Heible, Eds),

pp. 1-34. CRC Press, Inc., Boca Raton.

Holz, G. G., Rane, S. G., and Dunlap, K. (1986). GTP-binding proteins mediate

transmitter inhibition of voltage-dependent calcium channels. Nature. 319, 670-

672.

Horie, K., Obika., P., Foglar, R., and Tsujimoto, G. (1995). Selectivity of the

imidazoline alpha-adrenoceptor agonists (oxymetazoline and cirazoline) for

cloned alpha1-adrenoceptorsubtypes. Br. J. Pharmacol.116, 1611-1618.

Horn, J.P., and McAffee, D. A. (1980). Alpha-adrenergic inhibition of calcium­

dependent potentials in rat sympathetic neurones. J. Physiol. 301, 191-204.

Hirose, H., Maruyama, H., ltho, K., Koyama, K., Kida, K., and Saruta, T. (1993a).

Alpha-2 adrenergic agonism stimulates islet glucagon release from perfused rat

pancreas: possible involvement of alpha-2A adrenergic receptor subtype. Acta.

Endocrinol. 127, 279-283.

Hirose, H., Maruyama, H., ltho, K., Kida, K., Koyama, K., and Saruta, T. (1993b).

Effects of alpha2- and beta-adrenergic agonism on glucagon secretion from

perfused pancreata of normal and streptozocin-induced diabetic rats. Metabolism.

42, 1072-1076. 65

Hirose, H., Maruyama, H., ltho, K., Koyama, K. , Kido, K., and Saruta, T. (1993c).

Glucose-induced insulin secretion and alpha 2-adrenergic receptor subtypes. J.

Lab. Clin. Med. 121, 32-37.

Hirose, H., Seto, Y. Maruyama, H. Dan, K., Nakamura, K. Saruta, T. (1997). Effects

of alpha 2-adrenergic agonism, imidazolines, and G-protien on insulin secretion in

beta cells.

Hsu, W. H., and Hopper, D. L. (1986). Effect of yohimbine on amitraz-induced CNS

depression and bradycardia in dogs. J. Toxicol. Environ. Health. 18, 423-426.

Hsu, W. H., and Hummel, S. K. (1981). Xylazine-induced hyperglycemia in cattle: a

possible involvement of a 2-adrenergic receptors regulating insulin release.

Endocrinology. 109, 825-829.

Hsu, W. H., and Kakuk, T. J. (1984). Effect of amitraz and chlordimeform on heart

rate and pupil diameter: mediated by a 2-adrenceptors. Toxicol. Appl. Pharmacol.

73, 411-415.

Hsu, W. H., and Lu, Z. X. (1984). Amitraz-induced delay of gastrointestinal trasit in

mice: mediated by a 2-adrenergic receptors. Drug Develop. Res. 4, 655-660.

Hsu, W. H., LU, Z. X. , and Hembrough, F. B. (1986). Effect of amitraz on heart rate

and aortic blood pressure in conscious dogs: influence of atropine, prazosin,

tolazoline, and yohimbine. Toxicol. Appl. Pharmacol. 84, 418-422.

Hsu, W. H., and McNeel, S. V. (1985). Amitraz-induced prolongation of

gastrointestinal transit and bradycardia in dogs and their antagonism by

yohimbine: preliminary study. Drug Chem. Toxicol. 8, 239-253. 66

Hsu, W. H., and Schaffer, D. D. (1988). Effect of topical application of amitraz on

plasma glucose and insulin in dogs. Am. J. Vet. Res. 49, 130-131.

Hsu, W. H., Shaw, R. A., Schaffer, D. D. , crump, M. H., and Greer, M. H. (1987 ).

Further evidence to support the alpha 2-adrenergic nature of amitraz-induced

decrease in intestinal motility. Arch. Int. pharmacodyn. Ther. 286, 145-151 .

Hucht, A. M., Chelly, J., and Schmitt, H. (1981 ). Role of a1- and a2-adrenceptors in

the modulation of the baroreflex vagal bradycardia. Eur. J. Pharmacol. 71, 455-

461.

Hugnet, C., Buronfosse, F., Pineau, X., Cadore, J-L, Lorgue, G., and Berny, P. J.

(1996). Toxicity and kinetics of amitraz in dogs. Am. J. Vet. Res. 57, 1506-1510.

Ismail, N. A., El-Denshary, E. S., ldahl, L.A., Lindstrom, P. , sehlin, J., and Taljedal,

I. B. (1983). Effect of a-adrenoceptor agonists and antagonists on insulin

secretion, calcium uptake, rubidium efflux in mouse pancreatic islets. Acta.

Physiol. Scand. 118, 167-174.

Johansson, L. E., Nilsson, 0., and Olevall, 0. (1980). Amitraz-- (TAKTIC) for the

control of pig mange. Nord. Vet. Med. 32, 219-222.

Jorens, P. G., Zandijk, E., Belmans, L. , Schepens, P. J. , and Bossaert, L. L. (1997).

An unusual poisoning with the unusual pesticide amitraz. Hum. Exp. Toxicol.

Kagaruki, L. K. (1996). The efficacy of amitraz against cattle ticks in Tanzania.

Onderstepoort. J. Vet. Res. 63, 91-96.

Kashiwagi, A., Harano, Y. , Suzuki, M. , Kokima, H. , Harada, M., Nishio, Y. , and

Shigeta, Y. (1986). New a2-adrenergic blocker (DG-5128) improve insulin 67

secretion and in vivo glucose disposal in NIDDM patients. Diabetes. 35, 1085-

1089.

Kennedy, M. B. (1977). Amine metabolism: a different pathway in lobsters. Soc.

Neurosci. Abs. 3, 552.

Kennedy, M. B., (1978). Products of biogenic amine metabolism in the lobster:

sulphate conjugates. J. Neurochem. 30, 315-320.

Kennel, 0., Prince, C., and Garnier, R. (1996). Four cases of amitraz poisoning in

humans. Vet. Hum. Toxicol. 38, 28-30.

Kitagawa, H., and Walland , A. (1982). Augmentation of vagal reflex bradycardia by

central a 2-adrenceptors in the cat. Naunyn-Schmiedeberg Arch. Pharmacol. 321 ,

44-47.

Knowles, C. 0., and Benezet, H.J. (1981). Excretion balance, metabolic fate and

tissue residues following treatment of rats with amitraz and N'-(2,4-

dimethylphenyl)-methylformamidine. J. Environ. Sci. Health. 16, 547-555.

KobilKa, B. K., Matsui, H. Kobilka, T. S., Yang-Feng, T. L. , Francke, U., Caron, M.

G., Lefkowitz, R. J., and Regan , J.M. (1987). Cloning, sequencing and

expression of the gene encoding for the human platelet a 2-adrenergic receptor

subtypes. Science. 238, 650-656.

Koeiman, N. and Hsu, W. H. (1991). Interaction between amitraz and a2-

adrenceptors inhibits epinephrine-induced canine platelet aggregation. Arch. Int.

Pharmacodyn. Ther. 310, 56-65. 68

Koev, D., Borisova, A. M., Zlatareva, N., and Kirilov, G. (1989). Effect of

antihypertensive drugs on insulin secretion and efficacy. Vutr. Boles. 28, 23-28.

Lands, A. M., Arnold, A., McAuliff, J.P., Luduena, T. P., and Braun, T. G. (1967).

Differentiation of receptor systems activated by sympathomimetic amines.

Nature.214, 1316-1316.

Langer, S. Z. (1974). Presynaptic regulation of catecholamine release. Biochem.

Pharmacol. 23, 1793-1800.

Langer, S. Z. (1980). Presynaptic regulation of catecholamines. Pharmacol. Rev. 32,

337-364.

Lanier, S. M., Downing, S., Duzic, E., and Homey, C. J. (1991). Isolation of rat

genomic clones encoding subtypes of the alpha-2 adrenergic receptor. J. Biol.

Chem. 266, 10470-10478.

Lefowitz, R. J., Hoffman, B. B., and Tylaor, P. (1996). Neurotransmission. In The

Phamacological basis of Therapeutics, 9th ed. (J. G., hardman, L. E., Limbird, P.

B., Molinoff, R. W ., Ruddon, and A.G., Gilman, Eds.), pp. 126-129. McGraw-Hill,

New York.

Limbird, L. E. (1988). Receptors linked to inhibition of adenylate cyclase: additional

signaling mechanisms. FASEB. J. 2, 2686-2695.

Link, R., Daunt, D., Barsh, G., Chruscinski, A., and Kobilka, B. (1992). Cloning of

two mouse genes encoding a 2-adrenergic receptor subtypes and identification of

a single amino acid in the mouse a2c10 homolog responsible for an interspecies

variation in antagonist binding. Mol. Pharmacol. 42, 16-27. 69

Lomasney, J. W., Cotecchia, S., Lorenz, W., Leung, W. Y., Schwinn, D. A., Yang­

Feng, T. L., Brownstein, M., Lefkowitz, R. J., and Caron, M. G. (1991). Molecular

cloning and expression of the cDNA for the a 1-adrenergic receptor. J. Biol. Chem.

266, 6365-6369.

Lomasney, J. W., Lorenz, W., Allen, L. F., King, K., regan, J., Yang-Feng, T. L.,

Caron, M. G., and Lefkowitz, R. J. (1990). Expansion of the a 2-adrenergic

receptor family: Cloning and characterization of a human a 2-adrenergic receptor

subtype, the gene for which is located on chromosome 2. Proc. Natl. Acad. Sci.

U.S.A. 87, 5094-5098.

Lund, A. E., Shankland, D. L., Chinn, C., and Yim, G. K. W. (1978). Similar

cardiovascular toxicity of the pesticide chlordimeform and lidocaine. Toxicol. Appl.

Pharmacol. 44, 357-365.

MacNulty, E. E., McClue, S. J., Carr, I. C., Jess, T., Wakelam, M. J., and Milligan, G.

(1990). a 2-C10 adrenergic receptors expressed in Rat 1 fibroblasts can regulate

both adenylyl cyclase and phosphlipase D-mediated hydrolysis of

phosphatidylcholine by interacting with pertusis toxin-sensitive guanine

nucleotide-binding proteins. J. Biol. Chem. 267, 2149-2156.

Matsumura, F., and Beeman, R. W. (1976). Biochemical and physiological effects of

chlordimeform. Environ. Health Perspect. 14, 71-82.

McCall, R. B. , Schuette, M. R., Humphrey, S. J., et al. (1983). Evidence for a central

sympatoexcitatory action of alpha-2 adrenergic antagonist. J. Pharmacol. Exp.

Thre. 224, 501-507. 70

Medleau, L., and Willemes, T. (1995). Efficacy of daily amitraz on generalized

demodicosis in dogs. J. Small Anim. Prac. 36, 3-6.

Michel, A. D., Loury, D. N., and Whiting, R. L. (1989). Differences between a2

adrenoceptor in rat submaxillary gland and the a2A and a2B adrenoceptor

subtypes. Br. J. Pharmacol. 98, 890-897.

Mills, D. C. (1975). Initial biochemical responses of platelets to stimulation. Ciba.

Found Symp. 35, 153-167.

Minneman, K. P., Hedberg, A., and Molinoff, P. B. (1979). Comparison of beta 502-

adrenergic receptor subtypes in mammalian tissues. J. Pharmacol. Exp. Ther.

211, 508.

Mitra, M., Mahanta, S. K., Sen, S., Ghosh, C., and Hati, A. K. (1993). Sarcopties

scabiei in animals spreading to man. Trop. Georgr. Med. 45, 142-143.

Mojzisova, J., paulik, S., Bajova, V., and Baranova, D. (1977). {lmmunomodulatory

effect of levamisole and administration of amitraz in dogs with uncomplicated

generalized demodicosis]. Vet. Med. (Praha). 42, 307-3011.

Molinoff, P. B., Landsberg, L., and Axelrod, J. (1969). An enzymatic assay for

octopamine and other P-hydroxylated phenylethylamines. J. Pharmacol. Exp.

Thre. 170, 253-261.

Molinoff, P. B., and Axelrod, J. (1972). Distribution and turn over of octopamine in

tissues. J. Neurochem. 19, 157-163.

Morita, K., and North, R. A. (1981). Clonidine activates membrane potassium

conductance in myenteric neurones. Br. J. Pharmacol. 74, 419-428. 71

Morrow, A. N., Arnott, J. L., Heron, I. D., Koney, E. B., and Walker A. R. (1993). The

effect of tick control on the prevalence of dermatophilosis on indigenous cattle in

Ghana. Rev. Elev. Med. Vet. Pays. Trop. 46, 317-322.

Moser, V. C. , McPhail, R. C. (1985). Yohimbine attenuates the delayed lethality

induced in mice by amitraz, a formamidine pesticide. Toxicol. lett. 28, 99-104.

Muraki, T., Nakaki, T., and Kato, R. (1984). Predominance of a2-adrenoceptors in

porcine thyriod: biochemical and pharmacological correlations. Endocrinology.

114, 1645-1651.

Murphy, T. J., and Bylund, D. B. (1988). Characterization of alpha-2 adrenergic

receptors in the OK cell, an opossum kidney cell line. J. Pharmacol. Exp. Ther.

244, 571-578.

Nakaki, T., Nakadate, T., Yamamoto, S., and Kato, R. (1983). Inhibition of dibutyryl

cyclic AMP-induced insulin release by alpha-2 adrenergic stimulation. Life Sci. 32,

191-195.

Nathanson, J. A., and Greengard, P. (1973). Octopamine-sensetive adenylate 180,

cyclase: evidence for a biological role of octopamine in nervous tissue. Science.

308-310.

Nathanson, J. A. (1979). Octopamine receptor, adenosine 3' ,5' -monophoshate, and

neural control of firefly flashing. Science. 203, 65-68.

Niddam, R. Angel, I., Bidet, S., and Langer, S. Z. (1990). Pharmacological

characterization of alpha-2 adrenergic receptor subtype involved in the release of

insulin from isolated rat pancreatic islets. J. Pharmacol. Exp. Ther. 254, 883-887. 72

Noli, C., Van Der Horst, H. H., and Willemse T. (1996). Demodicosis in ferrets.

Vet.Q. 18, 28-31 .

Okada, S., Miyai, Y., Sato, K., Masaki, Y., Higuchi., T. et al., 1986; Ogino, Y., and

Ota, Z. (1986). Effect of clonidine on insulin secretion: a case report. J. Int. Med.

Res. 14, 299-302.

Orchard, I. (1982). Octopamine in insect: Neurotransmitter, neurohormone, and

neuromodulator. Can. J. Zool. 60, 659-669.

O'rourke, M. F., Iversen, J. W., Lomasney, J. W., and Bylund, D. B. (1994). Species

Ortholog of the Alpha-2A Adrenergic Receptor: The Pharmacological Properties of

the Bovine and Rat Receptors Differ from the Human and Porcine Receptors. J.

Pharmacol. Exp. Ther. 271, 735-740.

Ortiz-Alonso, F. J., Herman, W. H., Gertz, B. J., Williams, V. C., Smith, M. J., and

Halter, J.B. (1991). Effect of an oral a 2-adrenergic blocker (MK-912) on

pancreatic islet function in non insulin-dependent diabetes mellitus. Metabolism.

40, 1160-1167.

Pascoe, A. L., and Reynoldson, J. A. (1986). The cardiac effects of amitraz in the

guinea-pig in vivo and in vitro. Comp. Biochem. Physiol. 83, 413-417.

Pass M.A., and Mogg T. D. (1991). Effect of amitraz and its metabolites on intestinal

motility. Comp. Biochem. Physiol. 99, 169-172.

Pass, M.A., and Mogg, T. D. (1995). Pharmacokinetics and metabolism of amitraz

in ponies and sheep. J. Vet. Pharmacol. Ther. 18, 210-215.

Pass, M. A., and Seawright, A. A. (1982). Effect of amitraz on contractions of the

guinea-pig ileum in vitro. Comp. Biochem. Physiol. 73, 4190422. 73

Perez, D. M., Piascik, M. T., and Graham, R. M. (1991). Solution-phase library

screening for the identification of rare clones: isolation of an a 10-adrenergic

receptor cDNA. Mol. Pharmacol. 40, 876-883.

Piascik, M. T., Soltis, E. E., Piascik, M. M., and Macmillan, L.B. (1996). a­

Adrenoceptors and Vascular Regulation: Molecular, Pharmacological and clinical

Correlates. Pharmacol. Ther. 72, 215-241.

Porte, D., Girardier, L., Seydoux, J., Kanazawa, Y., and Posternak, J. et al. (1973).

Neural regulation of insulin secretion in the dog. J. Clin. Invest. 52, 210-214.

Porte, D., Graber, A. L., Kuzuya, T., Williams, R. (1966). The effect of epinephrine

on immunoreactive insulin levels in man. J. Clin. Invest. 45, 228-236.

Remaury, A. and Paris, H. (1992). The insulin-secreting cell line, RINm5F, express

alpha-20 adrenoceptor and nonadrenergic idazoxan-binding sites. J. Pharmacol.

Exp. Ther. 260, 417-426.

Reynoldson, J. A., and Cullen, L. K. (1996). Amitraz depresses cardiovascular

responses to bilateral carotid occlusion. J. Vet. Pharmacol. Ther. 19, 22-26.

Ristic, Z., Medleau, L., Paradis, M., and White-Weithers, N. E. (1995). lvermectin for

treatment of generalized demodicosis in dogs. J. Am. Vet. Med. Assoc. 207,

1308-1310.

Robert, M. C., and Argenzio, A. (1986). Effects of amitraz, several opiate derivatives

and anticholinergic agents on intestinal transit in ponies. Equine Vet. J. 18, 256-

260.

Robertson, P.R., Halter, J.B., and Porte, D. (1976). A role of a-adrenergic receptors

in abnormal insulin secretion in diabetes mellitus. J. Clin. Invest. 57, 791- 74

795.Regan, J. W., Kobilka, T. S., Yang-Feng, T. L., Caron, M. G., Lefkowitz, R. J.,

and Kobilka, B. K. Cloning and expression of a human kidney cDNA for an a2-

adrenergic receptor subtype. Proc. Natl Acad. Sci. U.S. A. 85, 6301-6305.

Roder, T., and Grweecke, M. (1990). Octopamine receptors in locust nervous tissue.

Biochemic. Pharmacol. 39, 1793-1797.

Roder, T. (1995). Pharmacology of the octopamine receptor from locust central

nervous tissue (OAR3). Br. J. Pharmacol. 114, 210-216.

Ros, J. J., and Aken, J. (1994). [Poisoning with amitraz, an agricultural anti­

ectoparasitic agent]. Ned. Tijdschr. Geneeskd. 138, 776-778.

Ruffolo, R.R., Nichols, A. J., Stadel, J.M., and Hieble, J.P. (1991). Structure and

function of a-adrenoceptors. Pharmacol. Rev. 43, 475-505.

Sabol, S. L., and Nirenberg, M. ( 1979). Regulation of adenylate cyclase of

neuroblastoma x glioma hybrid cells by a-adrenergic receptors. I. Inhibition of

adenylate cyclase mediated by a 2-receptors. J. Biol. Chem. 254, 1913-1920.

Saavedra, J. M., and Axelrod, J. (1973). Demonstration and distribution of

phenylethanolamine in brain and other tissues. Proc. Natl. Acad. Sci. U.S.A. 70,

769-772.

Saavedra, J. M., and Axelrod, J. (1976). Octopamine as a putative neurotransmitter.

Adv. Biochem. Psychopharmacol. 15, 95-110.

Saavedra, J. M. (1989). ~-phenylethylamine, phenylethanolamine, tyramine and

octopamine. Handb. Exp. Pharmacol. 90, 181-210. 75

Satoh, M., and Takayanagi, I. (1992). Identification and characterization of the alpha

20-adrenoceptor subtype in single cells prepared from guinea pig tracheal smooth

muscles. Jpn. J. Pharmacol. 60, 393-395.

Schaffer, D. D. , Hsu, W. H., and Hopper, D. L. (1990). The effects of yohimbine and

four other antagonists on amitraz-induced depression of shuttle avoidance

responses in dogs. Toxicol. Appl. Pharmacol. 104, 543-547.

Shin, D-H, and Hsu, W. H., Influence of the formamidine pesticide amitraz and its

metabolites of porcine myometrial contractility: involvement of a2-adrenceptors

and Ca2+ channels. Toxicol. Appl. Pharmacol. 128, 45-49.

Schmitt, H. , Fournadjive, G. , and Schmitt, H. (1970). Central and peripheral effects

of 2-(2,6-dimethylphenyl-amino)-4-H-5,6-dihydro-1,3-thiazin (Byer 1470) on the

sympathetic system. Eur. J. Pharmacol. 10, 230-238.

Schuit, F. C., and Pipeleers, D. G. (1986). Differences in adrenergic recognition by

pancreatic A and B cells. Science. 232, 875-877.

Schulz, A., and Hasselblatt, A. (1989). An insulin-releasing property of imidazoline

derivatives is not limited to compound that block alpha-adrenoceptors. Naunyn.

Schmiedebergs Arch. Pharmacol. 340, 321-327.

Schuntner, C. A., and Thompson, P. G. (1978). Metabolism of [14C] amitraz in

larvae of Boophilus micropuls. Aust. J. Biol. Sci. 31, 141-148.

Schwinn, D. A. , and Lomasney, J. N., Loreng, W., Szkult, P. J., Fremeau, R. T. ,

Yang-Feng, T. L. , Caron, M.G., letkowitz, R. J., and Cotecchia, S. (1990).

Molecular cloning and expression of cDNA for a novel a-adrenergic receptor

subtype. J. Biol. Chem. 265, 8183-8189. 76

Singh, G. J., Orchard, I., Loughton, 8. G. Octopamine-like actions of formamidines

on hormone secretion in the locust Locusta migratoria. Pest Biochem. Physiol. 16,

249-255.

Simonneaux, V., Ebadi, M., and Bylund, D. 8. (1991). Identification and

characterization of a 20-adrenergic receptors in bovine pineal gland. Mol.

Pharmacol. 40, 235-241.

Skoglund, G., Landquist, I., and Ahren, 8. (1986). Effect of a 1- and a 2-adrenoceptor

stimulation and blocked on insulin levels in the mouse. Pancreas. 1, 412-420.

Smith, 8. E., Hsu, W. H., and Yang, P-C. (1990). Amitraz-induced glucose

intolerance in rats: antagonism by yohimbine but not by prazosin. Arch. Toxicol.

64, 680-683.

Smith, P.H., and Porte, D. (1976). Neuropharmacology of the pancreatic islets.

Annu. Rev. Pharmacol. Toxicol. 16, 269-285.

Strake, K. (1981). a-adrenoceptors subclassification. Rev. Physiol. Biochem.

Pharmacol. 88, 199-236.

Starke, K. (1980). Presynaptic receptors. Ann. Rev. Pharmacol. Toxicol. 21, 7-30.

Svensson, S. P., Bailely, T. J., Porter, A. C., Richman, J. G., and Regan, J. W.

(1996). Heterologous expression of the cloned guinea pig a 2A, a 28, a 2C

adrenoceptor subtypes: Radioligand binding and functional coupling to a cAMP

responsive receptor gene. Biochem. Pharmacol. 51, 291-300. 77

Sweatt, J. D., Connolly, T. M., Cragoe, E. J., and Limbired, L. E. (1986). Evidence

that Na+/ H+ exchange regulates receptor-mediated phospholipase A2 activation in

human platelets. J. Biol. Chem. 261, 8667-8673.

Sweatt, J. D., Johnson, S. L., Cragoe, E. J., and Limbired, L. E. (1985). Inhibitors of

Na+/ H+ exchange block stimulus-provoked arachidonic acid release in human

platelets. J. Biol. Chem. 260, 12910-12919.

Tominaga, M., Maruyama, H., Vasko, M. R., Baetens, D., Orci, L. , and Hunger, R. H.

(1987). Morphological and functional changes in sympathetic nerve relationships

with pancreatic a-cells after destruction of p-cells in rats. Diabetes. 36, 365-373.

Tudek, B., Gajewska, J., Szczypka, M., and Rahden-staron, I. (1988). Screening for

genotoxic activity of amitraz with short-term bacterial assays. Mutat. Res. 204,

585-591.

Van Zwieten, P.A., and Timmermans, P. B. (1983). Cardiovascular a 2-receptors. J.

Mol. Cell Cardiol. 15, 717-733.

Waeber, G. Gomez, F., Chaubert, P. , Temler, E., Chapuis, G., Boulat, 0., Nicod, P.,

Haefliger, J. A. (1997). In vivo and in vitro effects of somatostatin and insulin on

glucagon release in a human glucagonoma. Cline. Endocrinol. 46, 637-642.

Wang, C. M., Varahashi, T., and Fukami, J. (1975). Mechanism of neuromuscular

block by chlordimeform. Pest. Biochem. Physiol. 5, 119-125.

Weinshank, R. L., Zgombick, J. M., Macchi, M., Adham, N., Lightblau, H., Branchek,

T. A., and Hartig, P.R. (1990). Cloning expression and pharmacological 78

characterization of a human a 28-adrenergic receptor. Mol. Pharmacol. 35, 681-

688.

Woodcock, E. A., and Johnston, C. I. (1982). Selective inhibition by epinephrine of

parathyroid hormone-stimulated adenylate cyclase in rat renal cortex. Am. J.

Physiol. 242, F721-F726.,

Wright, F. C., Guillot, F. S., and George, J. E. (1988). Efficacy of acaricides against

chorioptic mange of goats. Am. J. Vet. Res. 49, 903-904.

Wu, P. H., and Boulton, A. A. (1979). N-Acylation of tyramines: purification and

characterization of an arylamine N-acetyltransferase from rat brain and liver. Can.

J. Biochem. 57, 1204-1209.

Yamazaki, S., Katada, T., and Ui, M. (1982). a2-Adrenergic inhibition of insulin

secretion via interference with cyclic AMP generation in rat pancreatic islets. Mol.

Pharmacol. 21, 648-653.

Yang, C., and Hsu W. H. (1995). Stimulatory effect of bradykinin on insulin release

from the perfused rat pancreas. Am. J. Physiol. 288, E1027-E1030.

Yang, H. Y., and Neff, N. H. ( 1976). Brain N -acetyltransferase: substrate specificity,

distribution and comparison with enzyme activity from other tissues.

Neuropharmacology. 15, 561-564.

Zeng, D., Harrison, J. K., D'angelo, D. D. , Barber, C. M., Tucker, A. L., Lu, Z., and

Lynch, K. R. (1990). Molecular characterization of a rat a28-adrenergic receptor.

Proc. Natl. Acad. Sci. U.S.A. 87, 3102-3106. 79

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude and deepest appreciation to my

major professor, Dr. Walter H. Hsu, for his guidance, support, and continues help in

solving the problems I faced during my master program at Iowa state university. will be forever grateful for his efforts. I would also like to thank Dr. Franklin A.

Ahrens and Dr. David L. Hopper for their serving on my POS committee and for their valuable time read ing and correcting this thesis. My thanks are extended to my fellow graduate students Sirintorn Yibchokanun (Lek), Jeff Makowski, Jing Ding, and

Henrique Cheng. Also I wish to thank Mr. Laverene Escher for his technical

assistance and friendship. The love and concern of every member of my family will

always be remembered. My feeling of responsibility toward them is one of the most

important motivations for me in every step of my academic career. Lastly, I wish to thank all my real friends for their sincere concern and support.