The Effects of Local Intravascular Infusion of Bradykinin on Submaxillary Saliva in the Cat John A

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1971 The effects of local intravascular infusion of bradykinin on submaxillary saliva in the cat John A. Patti Yale University

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THE EFFECTS OF LOCAL INTRAVASCULAR

INFUSION OF BRADYKININ ON

SUBMAXILLARY SALIVA IN THE CAT

John A„ Patti

A Thesis

Presented to the Faculty of the School of Medicine

Yale University

In Partial Fulfillment of the Requirement

for the Degree of

Doctor of Medicine

Department of Pediatrics

1971 .

O -£> I would like to acknowledge the support and counsel given by Dr0 Lewis E. Gibson not only during the course of this investiga¬ tion but whenever they were sought. . : o " _ * o : ~ c =>. nw I or, i . ■ e. . o ■

-.R 'i ~ . j - O c r-c ~ - o C. , O =\ oj Abstract

The influence of bradykinin on submaxillary saliva was investigated in an attempt to elucidate the pathogenesis of cystic fibrosis,, Using contralateral glands as controls, submaxillary glands of cats were perfused with synthetic bradykinin of a concentration in excess of that found in human plasma. Patterns of flow rate, concentrations and excretions of calcium, sodium, potassium and chloride were determined. These patterns exhibited no resemblance to those found in patients with cystic fibrosis. The results of this investigation appear to indicate that the clue to the pathogenesis of cystic fibrosis does not lie in the kallikrein- kinin system. Alternate theories of the basic defect in cystic fibrosis are discussed and the direction of further investigation is indicated. Sj j. - o o : - . _ -

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i Introduction

The influence of bradykinin on submaxillary saliva was investigated

in an attempt to elucidate the pathogenesis of cystic fibrosis (C. F,).

C. F0 is an inherited disorder that affects many and perhaps all exocrine 1 glands . Well defined abnormalities have been observed in both sweat 2 and saliva, as well as in pancreatic secretions . The kallikrein kinin

system is thought to be involved in the normal function of these exocrine 3 glands. Fox and Hilton have demonstrated that vasodilatation occurring in the human forearm on indirect heating has several features in common with salivary gland vasodilatation, and that sweat gland activity leads to the appearance of kallikrein in the sweat and to the formation of bradykinin

in the skin. Bradykinin production has been demonstrated in the active 4 pancreas , and this vasoactive peptide is released in shock-producing 5 quantities in acute experimental pancreatitis . Electron microscopic and histochemical localization studies in guinea pigs and cats have demonstrated

electron dense and PAS-positive kallikrein-containing granules in both the 6-8 pancreas and submaxillary glands

There is some controversy regarding the role of the kinin system in the normal function of the salivary glands. Salivary kallikrein was first 9 described in 1936 by Werle and Roden , and during the same year, Ungar 10 and Parrot suggested that salivary kallikrein is released during salivary

secretion and that it is the mediator of the atropine-resistant vasodilatation

produced by chorda nerve stimulation in the submaxillary gland. This theory • ;

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11-13 is supported by the work of Hilton and Lewis , who showed that kallikrein was released from the submaxillary gland of the cat after

stimulation of the chorda nerve, and that this release was not affected by administration of atropinec 14-19 Criticism of this theory has arisen from recent studies 16 Bhoola et al have shown that in a submaxillary gland perfused with a kininogen-free fluid, maximal vasodilatation can be achieved by chorda 18 stimulation. Skinner and Webster demonstrated that perfusion of the cat's submaxillary gland with carboxypeptidase B, a kininase, completely blocked the vasodilatation produced by bradykinin infusion but had no effect 19 on vasodilatation produced by chorda stimulation. Beilenson et al found that sympathetic stimulation produces a higher concentration and output of kallikrein from the submaxillary gland than does parasympathetic stimula¬ tion. They also found that glands depleted of kallikrein responded to chorda stimulation with normal, atropine resistant vasodilatation, and thus suggested that the kallikrein-kinin system does not play a significant role in submaxillary gland vasodilatation.

More recent studies lend support to the theory of kinin-mediated vaso- 5 dilatation in exocrine glands. Popieraitis and Thompson demonstrated that intra-arterial infusion of bradykinin led to an increase in both the volume 20 and blood content of the pancreas. Gautvik et al have shown that the level of kininogenase in the venous effluent of the cat's submaxillary gland decreases when the gland is activated either by chorda nerve or acetycholine

stimulation and that this increase is not affected by atropine. Further « •' • o c . x c : o £ " c i

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21 studies by Gautvik indicated the presence of two vasodilator mechanisms in the submaxillary gland. The first is a rapid vasodilatation induced by chorda stimulation. The second, later phase of vasodilatation strongly suggests the formation of kinins secondary to activation of the gland.

Despite existing controversy, the kinin system appears to play some role, although not well-defined, in normal salivary gland function.

The role of any specific substance, however, in the pathogenesis of 22 cystic fibrosis is quite unclear. Observations made by Spock et al indicate that there is an abnormal serum factor in patients with C. F. which is not present in normal children and is present in small concentra¬ tions in parents of children with C. F. This factor causes an asymmetrical ciliary beat in explanted human and animal respiratory-tract epithelium. 23 It has also been found to inhibit ciliary motion in oysters . The factor is present in the euglobulin fraction of serum, and activity has been 22 24 demonstrated in the 19S macroglobulins . Haverback et al have isolated a 19S macroglobulin in plasma which binds trypsin in a manner which pro¬ tects its esterolytic activities from inhibition, and have shown that a mechanism exists in plasma for the binding or conjugation of kallikreins to this macroglobulin. They have also shown that the amount of trypsin that can be bound by serum of C„ F. patients was twice that of control hospitalized patients. Using a modified Schultz-Dale assay apparatus, 25 Lieberman and Littenberg showed that saliva from patients with C. F, had a significantly greater kallikrein content than saliva from controls. s r •o. oil c ■ - c c so.-: - • :-£CX i.i hr .; s y ' : si, I

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We therefore originated the hypothesis that the serum factor in cystic fibrosis is responsible for binding and protecting kallikrein and that this

excess available kallikrein produces excess amounts of bradykinin in

exocrine glands. A problem arose concerning the best method to test this hypothesis. Direct infusion of serum of C. F. patients into an animal

could not be undertaken because kallikreins of one species are notably 16, 26 ineffective in releasing kinins in different species

Submaxillary glands of patients with C. F secrete saliva with different

electrolyte concentrations and at different flow rates than those found in 27 normal children. Mandel et al have shown that C. F, submaxillary

saliva contains significantly higher concentrations of calcium, sodium and chloride, higher potassium concentrations and lower flow rates than 28, 29 saliva from normal children. Other investigators have been able to demonstrate significant elevations only of calcium in C.F. submaxillary 35 saliva. Although serum calcium concentrations are normal , elevations 30, 31 of calcium concentration have also been found in parotid saliva , 32 33 34 35 sweat , tracheobronchial secretions , meconium and duodenal fluid of patients with C.F.

The half life of synthetic bradykinin in blood is 17 seconds and brady- 36 kinin is inactivated in the lung „ This investigation was therefore designed to use one submaxillary gland of the cat as a control, and to-perfuse the other submaxillary gland with synthetic bradykinin in an attempt to show a significant elevation of salivary calcium concentrations and to reproduce the electrolyte pattern found in cystic fibrosis. c o

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Materials and Methods

Mature cats weighing between 2. 3 kg and 4. 3 kg were anesthetized with intravenous Nembutal (10 mg/kg)„ An intravenous catheter was placed in the left fore leg using a polyethelene tube (PE 50) through which maintenence fluids (5% dextrose in 1/2 normal saline) were delivered at a rate of 0. 144 ml/kg/min by means of a constant in¬ fusion pump, and through which Nembutal was administered as necessary to maintain surgical levels of anesthesia. A midline incision was made in the neck, through which a tracheotomy was performed and a tracheos¬ tomy tube inserted. The right common carotid artery was then isolated and cannulated with polyethelene tubing (PE 10) through which fluids were delivered at a rate of 0. 057 ml/kg/min by means of a constant infusion pump.

This catheter offered little obstruction to arterial flow. The mouth was then held open and the papillae containing the terminal arborizations of both left and right submaxillary ducts were excised, thus exposing the main sub¬ maxillary ducts. These ducts were cannulated with the attenuated ends of equal lengths of polyethelene tubing (PE 90). Pilocarpine was infused at a constant rate of 5.76 mg/kg/min through the intravenous fore leg catheter.

After free flow of saliva was established for 30 minutes and a steady state achieved, collection of saliva into tared vials was begun. Collection periods were 5 minutes each and were grouped into 3 longer periods of

30 minutes each. During the first 30 minute period, hereafter referred to as T^ for the cannulated test side and for the control side, normal O P • . >■ \, . c '• ].

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saline was infused into the cannulated common carotid artery. During the

second 30 minute period (C£ and T^), synthetic bradykinin (Sandoz) was infused at a constant rate of 0.456 pg/kg/min into the cannulated artery.

During the third 30 minute period (C3 and T^), normal saline was infused into the cannulated carotid. The arterial supply to the contralateral sub¬ maxillary gland was unaltered, and saliva from that gland served as a control.

Vials were re-weighed immediately after collection and, assuming a specific gravity of 1. 000 for saliva, flow rates were calculated. Each sample was then diluted with 5 ml. distilled water and analyses of calcium,

sodium, potassium and chloride were performed. Calcium concentrations were determined using a Perkin-Elmer Atomic Absorption Spectrophoto¬ meter Model 303 with distilled water as a blank. Chloride analyses were performed on a Cotlove chloridometer, and sodium and potassium deter¬ minations were made with a flame photometer against a lithium blank.

Duplicate analyses were performed which correlated closely with each other.

Res ult s

Experiments were conducted on 15 animals and the results obtained from 7 of those animals are presented here. Reasons for the inability to include results from the remaining 8 animals are as follows: anesthetic death (2 animals), inability to cannulate submaxillary ducts (2 animals), intermittent blockage of submaxillary ducts (3 animals) and blockage of

carotid artery cannula (1 animal).

Parameters measured in these experiments were salivary flow rates, concentrations of calcium, sodium, potassium and chloride, and excretion

rates of these electrolytes. Results from each 5 minute sample were av¬

eraged to obtain mean values for C^, C^j T|, T^, and for each animal. These values were grouped, a mean for all cats obtained and

standard deviations calculated. Results are presented in figs 1-9 and tables 1-3. Changes in all parameters (C^-C^), (C^-£>2)* (T2~T^) an<^

(T^-T^) were calculated for each animal. These values were grouped, a mean for all cats obtained and standard deviations calculated. Results are presented in tables 4-6. Although the data presented indicate trends of increasing or decreasing mean values, not all animals followed the trend for a given parameter.

The effects of bradykinin infusion were interpreted as the mean change of test parameters from steady state levels, compared to the mean change of control parameters during the same collection periods (T^-T^) - (C^-C^)

Likewise, the effects of cessation of bradykinin infusion were interpreted

as the mean change of parameters from levels during bradykinin infusion

compared to the mean change of control parameters during the same

collection periods (T^-T^) - (C^-C-^) . The degree of statistical signi¬ ficance of test changes compared to control changes was determined by the

Students "T" test.

Salivary flow rates (fig. 1, table 1) of control glands showed a gradual

decrease over the total collection time from a mean steady state rate of

0.243 ml/min to a mean rate of 0.195 ml/min. Test glands showed a de¬

crease in flow rate from 0.218 ml/min to 0. 208 ml/min with infusion of bradykinin, and a further decrease from 0.208 ml/min to 0. 188 ml/min with cessation of bradykinin infusion. During infusion of bradykinin there was a decrease in flow rate of 0.004 ml/min (table 4) greater than the decrease in controls (p>0.50). Flow rate of experimental saliva after cessation of bradykinin infusion decreased by 0. 014 ml/min less than the decrease in control rate during the same collection period (p>0.50).

Mean calcium concentrations (fig, 2,table 2 ) of control saliva showed an initial increase from 1. 10 mEq/L to 1.49 mEq/L, followed by a slight decrease to 1.43 mEq/L. Experimental saliva showed an increase from a mean steady state calcium concentration of 0.86 mEq/L to 1.06 mEq/L with infusion of bradykinin, followed by a further increase to a mean concen¬ tration of 1.62 mEq/L after cessation of bradykinin infusion. During infusion of bradykinin there was an increase in calcium concentration of 0. 19 mEq/L less than the increase in control saliva (p>. 50) (table 5). Calcium concen¬ tration of experimental saliva after cessation of bradykinin increased by ::jr ilfxx?.'vi>J5*xi si :Q- - ■ :: •“ ■=> •

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0. 62 mEq/L compared to the change in control calcium concentration

during the same collection period (p >. 10). Neither of these increases were statistically significant. Patterns of control and experimental rates

of calcium excretion were similar to each other (fig. 3, table 3) with an

initial increase in excretion rate followed by a slight decrease. Mean

changes were insignificant (table 6), showing that during infusion of brady-

kinin there was an increase in calcium excretion of 0. 017 pEq/min greater than the increase seen in control saliva (p >. 50). Calcium excretion of

experimental saliva after cessation of bradykinin infusion decreased by

0.018 [iEq/min less than the decrease in control calcium excretion during the same collection period (p > . 40).

The pattern of sodium concentration and excretion in experimental

saliva was not significantly different from that of control saliva. Sodium

concentrations of both control and experimental saliva showed a steady but insignificant increase over the entire collection period (fig. 4, table 2) from 33.2 mEq/L to 67.7 mEq/L in controls, and from 20.2 mEq/L to

42.4 mEq/L in experimental saliva. During infusion of bradykinin there was mean increase of 9. 5 mEq/L less in experimental saliva than the increase

seen in control saliva (p > 0.50) (table 5). Sodium excretions (fig. 5, table 3)

of both control and experimental saliva also showed a steady increase over the whole collection period from 4. 55 pEq/min to 8. 47 pEq/min in controls

and from 4.07 |fEq/min to 7.32 pEq/min in experimental saliva. During

infusion of bradykinin there was a mean increase of 0.94 pEq/min less in

experimental saliva than the increase seen in control saliva (p >0. 50), and T \ oi j r o: C

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after cessation of bradykinin infusion there was an increase of 0.29 Eq/min more in experimental saliva than the increase seen in control saliva (p>0. 50)

(table 6)„

Potassium concentrations (fig 6, table 2) in control saliva rose from

9.6 mEq/L to 12.9 mEq/L over the entire collection period, whereas experimental salivary concentrations rose from 10. 1 mEq/L to 11.0 mEq/L during the infusion of bradykinin and then fell to 10.6 mEq/L after cessation of bradykinin infusion. The decrease in potassium concentration after cessation of bradykinin infusion is more apparent than real since there is considerable overlap of individual control and experimental values. During the infusion of bradykinin there was an increase of 0.6 mEq/L less in experimental saliva than the increase observed in control saliva (p>0. 20).

Potassium concentration of experimental saliva after cessation of bradykinin infusion decreased by 2.2 mEq/L compared to the change in control potassium concentration during the same collection period (p>0„ 05) (table 5).

Potassium excretions (fig. 7, table 3) exhibited a small but insignificant increase from 1.84 n Eq/min to 1.94 M-Eq/min in control saliva followed by a decrease to 1.81 PEq/min, and showed a decrease from 2.40 MEq/min to 1

2. 30 fjEq/min in experimental saliva with infusion of bradykinin, followed by a further decrease to 2. 10 pEq/min in experimental saliva after cessation of bradykinin infusion. Bradykinin infusion thus was accompanied by a mean decrease in potassium excretion of 0. 20 pEq/min compared to the change in control saliva during the same collection period (p>0o30). Potassium excretion of experimental saliva after cessation of bradykinin infusion decreased by [■ -.W .

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same collection period (p>0o 50) (table 6), Neither of these decreases are significant.

Chloride concentration (fig. 8, table 2) in control saliva showed a

rise from 6. 75 mEq/L to 9. 78 mEq/L, which was paralleled by a rise from 7.79 mEq/L to 10.9 mEq/L in experimental saliva with infusion

of bradykinin. Control chloride concentrations then decreased slightly to 9.23 mEq/L but experimental concentrations continued to rise to

17. 1 mEq/L after cessation of bradykinin infusion. Infusion of brady¬

kinin was thus associated with an insignificant (p>0»50) mean increase

in chloride concentration of 0.08 mEq/L greater than the rise in control

values. Chloride concentration of experimental saliva after cessation

of bradykinin infusion increased by 6.75 mEq/L compared to the change in

control values during the same collection period (table 5). The latter in¬

crease,although large, is statistically insignificant (p> 0. 10). Chloride

excretions (fig. 9, table 3) of control saliva showed a steady decrease over

the entire collection period from 1. 84 MEq/min to 1.49 M-Eq/min. Experi¬

mental values increased from 2. 37 pEq/min to 3. 22 pEq/min with infusion

of bradykinin and continued to rise to 3. 39 M-Eq/min after cessation of

bradykinin infusion. During infusion of bradykinin there was an increase in

chloride excretion of 0.91 MEq/min compared to the change in control saliva

(p>0.20) chloride excretion after cessation of bradykinin infusion increased

by 0.46 pEq/min compared to the change in control chloride excretion during

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Fig. 3 Calcium Excretion

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Sodium Concentration (mEq/I. i 1 Collection Period; 2 Sodium Concentration 0~ Experimental x— Control Figo 4 3 i^lPk [C77 k-P~rrj 15

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Sodium Excretion ( g Eq/min) 1 Collect ion Periods 2 Sodium Excretion O =Experimental c ■-Control Fig. 5 3 16

15 - 5 -- s 11!_!._j (ya | r"H [C:i ' rl:. J 1 Potassium Concentration (mEq/L) PfH jTiqcq ;:u itirctil ±±xt Lrr.ti.ii; Ertcrr 1 ;:J Isj: C,j Wit ±n*i tj ’Cl ■rfL4: HTti 1 l ESS m±m i n-ggp i r ;.::K Collection Periods t-—r~ z m Potas siumConeentration V =Control U= Experimental i rf'•tti±'4- tricri' tccIecC:ji jlHEa Fig. 6 ■ LITTVCCC -PqE~ -:Py;-Z.L ::cifr;:c . :::~TT^:.rr.rt - :—U-f- ~r_ ■nrr.tT Tffrrfffiri .■“cerr.r.:' j_ "'-FpPrj; . Hh*rFt icintcnitip CPC -E •i FrrrH* 1 ;fh 17 w- 18

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Fig. 8 Chloride Concentration \/ — Control 0 = Experimental

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Collection Periods

1 2

Fig. 9 Chloride Excretion \? ~ Control 0=Experimental

0 Chloride Excretion ( M-Eq/min)

Table 1

Mean Salivary Flow Rates

Mean S. D. Mean S. D„

0o 243 0o 148 0.218 0. 021 C1 Ti

0. 229 0, 174 0. 208 0. 093 C2 T2

0. 195 0. 138 0. 188 0. 097 C3 T3

values are expressed in ml/min

Table 2

Mean Electrolyte Concentrations

Mean S0 D. Mean So Do

1. 10 0. 66 0. 86 0. 26 ci T1 Ca 1.49 1.05 1.06 0o 57 C2 T2

1.43 0. 69 1.62 0. 83 C3 T3

ci 33. 2 44. 1 T1 20. 2 18 o 1

Na C2 55. 4 46 o 6 T2 32. 8 16o 7

C3 67. 7 52o 5 T3 42. 4 17o 3

ci 9. 6 3. 1 Tl 10o 1 lo 1 K C2 11. 1 4. 2 T2 11.0 2. 6

c3 12. 9 5. 7 T3 10o 6 1 o 9

ci 6. 75 2. 77 Tl 7. 79 6o 62

Cl C2 9. 78 6o 93 T2 10o 89 lo. 12

C3 9.. 23 8. 00 T3 17. 10 IQ. 61

values are expressed in mEq/L ~ ,r

lo 22

Table 3

Mean Electrolyte Excretions

Mean So D. Mean SoD.

0o 184 0o 101 0o 192 0. 073 ci T1

Ca 0. 250 0. 206 0. 275 0o 164 C2 T2

C3 0o 226 0. 188 T3 0o 269 0o 150

4. 57 3o 97 40 07 3. 17 ci T1 Na 7. 03 6. 94 5o 59 3„ 03 C2 T2

C3 8, 47 7. 60 t3 7„ 32 5o 36

1.84 0o 75 2. 40 0. 45 C1 T1

K 1.94 0. 98 2. 30 1.04 C2 T2

C3 1.81 1.01 T3 2. 10 0. 24

1.84 lo 40 2„ 37 lo 78 C1 ti

Cl 1.78 0. 78 3. 22 3. 55 C2 T2

C3 1.49 I. 08 T3 3.39 3. 39

values are expressed in iaEq/min cj - ' -: j 23

Table 4

Mean Salivary Flow Rate Changes

C2~C1 T2-Tl C3-C2 T3-T2

Mean -Oo 014 -Oo 010 -0o 034 -0o 020

S„ D„ 0. 045 Oo 079 0o 083 0o 048

values are expressed in ml/min

Table 5

Mean Electrolyte Concentration Changes

T -T T,-T C2-°l 2 1 C3-C2 3 2 vO o o 1 Mean + 0. 39 + 0o 20 o + 0o 56 Ca SoDo lo 00 0o 12 Oo 44 0. 54

Mean + 22. 1 + 12. 6 + 1 2„ 4 + 9. 8 Na SoDo 10o 1 8. 6 6. 5 18. 3

Mean + 1.5 + 0o 9 + 1.8 - 0. 4 K SoD. 1. 1 1. 5 1.6 0o 7

Mean + 30 03 + 3. 11 - 0o 55 + 6o 20 Cl

So D. 5„ 91 5o 24 lo 67 9o 59

values are expressed in mEq/L . cl .•? ul> i ,S‘-

.... ~ -

s l

. 24

Table 6

Mean Electrolyte Excretion Changes

Cl, cz Ti. T2 c2> C3 t2>

Mean + 0. 066 + 0.083 - 0.024 - 0.006 Ca

S. D. 0. 163 0. 108 0. 037 0. 056

Mean + 2. 46 + 1. 52 + 1.44 + 1.73 Na

S0 D. 3. 44 1. 76 2. 09 4. 28

+ 0. 10 - 0. 10 - 0. 13 - 0.20 K Mean

0. 25 0. 85 S„ D. 0. 09 0,89 + 1—' o Mean - 0.06 + 0. 85 - 0. 29 o Cl

S„ D. 0. 79 2. 44 0. 97 1.98

values are expressed inpEq/min oh ' 25

Dis cus sion

It was thought that if exocrine glands in patients with C. F» were chronically exposed to high concentrations of bradykinin, an attempt to duplicate this situation in an acute experiment could be made by using 37 very high local concentrations of bradykinin. Borcklehurst and Zeitlin have determined that the free plasma kinin level in man is 2.8 ng/ml.

Based on an estimate of unilateral carotid blood flow (7. 5% of cardiac output) and an estimated cardiac output (71 ml/min/kg) coupled with the known concentration and rate of infusion of the synthetic bradykinin, the calculated concentration of bradykinin in the plasma flowing through the experimental carotid artery was approximately 0. llOgg/ml. This concen¬ tration was decided upon after preliminary experiments using 2. 5% and

25% of this concentration failed to yield positive results. Neither the infusion of this large excess (0. 110 |jg/ml) of bradykinin nor the cessation of infusion resulted in any significant difference in the pattern of flow rate, electrolyte concentrations or excretions when compared to control patterns of these parameters.

There is a possibility, with such a large dose, that not all of the in¬ fused bradykinin was inactivated in the lungs and that some bradykinin reached the control gland. If this were indeed the case, one would expect any effects of bradykinin to occur in both glands. These effects would be observable as a significant change in parameters from values in collection period 1 to higher or lower values in collection period 2. P values for any changes occurring in either gland between these two collection periods

were all greater than 0.20„ Thus, even if bradykinin did reach the control gland, it did not result in a significant change in any of the para¬ meters measured.

Both control and experimental electrolyte concentrations increased steadily with time in the presence of a steadily decreasing flow rate. 38,39 These observations agree with those of some investigators but dis- 40, 41 38 39 agree with those of others . Both Dreisbach and Neilsen have demonstrated an inverse relationship between calcium concentration and flow rate in submaxillary saliva after pilocarpine stimulation. Young and 40 41 Schogel and Petersen and Poulsen , however, have shown increases in sodium and potassium concentrations with an increase in flow rate. A possible explanation for our observations is that dehydration may have occurred in our animals secondary to constant pilocarpine infusion.

Another interesting observation is that infusion of bradykinin did not increase salivary flow rates. If kinin formation is truly responsible for a significant degree of vasodilatation in the salivary gland, one would expect an increase in flow rate to occur with infusion of bradykinin. Our inability to demonstrate this increase would seem to support the theory of 7 Heap and Bhoola who suggest that the PAS-positive kallikrein-containing granules present in the submaxillary glands are secreted into the acinar or duct lumen rather than into the extracellular tissue space, thus indi¬ cating that kallikrein performs an exocrine rather than a local endocrine role in the submaxillary gland. o ■ o r r o C • V

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The large standard deviations and considerable overlap of control and experimental values in this investigation do not allow one to speculate further on possible causes of the observed changes because these changes may be more apparent than real.

Although the kallikrein-kinin system has been implicated in many disease processes, including pancreatitis, asthma, thermal edema, dump¬ ing syndrome, migraine, arthritides, and shock reactions, it has been shown to be significantly involved only in carcinoid syndrome and hereditary 42, 43 angioedema . The results of this investigation indicate that the local infusion of high concentrations of bradykinin does not alter certain para¬ meters of submaxillary saliva and does not reproduce the patterns of these parameters found in children with cystic fibrosis, thus casting some doubt on the role of the kallikrein-kinin system in the pathogenesis of that disease. 44 Mangos et al have demonstrated that retrograde perfusion of the ratIs parotid gland with saliva from C. F. patients decreased the rate of sodium reabsorption, whereas perfusion with saline or normal saliva did not affect this rate. They postulate the existance of a sodium transport inhibitory 45 factor in the saliva of patients with C.F. Gibson et al , however, feel that a defective mucus water barrier is present in C.F. exocrine ducts, thus leading to excess passive water reabsorption and resulting in hypertonic and viscid exocrine secretions. They have shown that the rate of flow of isotonic

~~saline through the non-dialyzable fraction of C.F. submaxillary saliva was greater than the rate of flow through similar material from normal children. ,-c od • o - •; o r s o _■ „ .s..~~

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They have also demonstrated that when calcium is added to normal submaxillary saliva, flow rates through the non-dialyzable fraction were increased to the levels found in C. F. saliva. 46 Gugler et al have investigated the role of calcium in submaxillary saliva of C. F0 patients. They have found that normal saliva is clear and demonstrates a constant electrophoretic pattern, whereas C. F. saliva is turbid and its electrophoretic pattern differs quantitatively though not qualitatively from the pattern seen in normal saliva. When a calcium chloride solution was added to normal saliva, the saliva became turbid and exhibited an electrophoretic pattern similar to that seen in C. F. saliva.

When EDTA was added to C. F. saliva, the saliva became clear and the electrophoretic pattern resembled that seen in normal saliva. They thus suggested that the excess calcium in C. F. exocrine secretions forms a reversible precipitate with mucus glycoproteins. This precipitate could result in the excess water reabsorption postulated by Gibson et al.

~~Clarification of these theories awaits further investigation of the ability of C. F. serum to produce increased calcium concentrations in exocrine secretions. : c, o o~~

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~~References~~

~~I. di Sant’Agnese, P. A. and Talamo, R. C. Pathogenesis and pathophysiology of cystic fibrosis of the pancreas. NEJM 277: 24s 25, 26, 1287, 1344, 1399 1967.~~

~~2„ Nelson, W. E. Textbook of Pediatrics, pp. 856-867 W. B. Saunders, Philadelphia, 1969.~~

~~3. Fox, R. H. and Hilton, S0 Ma Bradykinin formation in human skin as a factor in heat vasodilatation. J. Physiol 142: 219, 1958.~~

~~4. Hilton, S. M., and Jones, M. Plasma kinin and functional vasodila tation in the pancreas. Ja Physiol 165: 35P, 1963.~~

~~5. Popieraitis, A. S. and Thompson, A, G. The site of bradykinin re lease in acute experimental pancreatitis. Arch. Surg (Chicago) 98: 73, 1969.~~

~~6. Bhoola, K. D0 and Heap, P. F. Electron microscopy and histo¬ chemistry of isolated kallikrein granules. Brit. J. Pharmacol. 38: 435P, 1970.~~

~~7. Heap, P» F. and Bhoola, K, D. Histochemical localization of kallikrein granules in the submaxillary gland of the guinea pig. J. Anat. 105: 525, 1969.~~

~~8. Bhoola, K. D. and Dorey, G. Isolation of kallikrein containing granules from the pancreas and submaxillary gland of the cat. J. Physiol. 203: 59P, 1969.~~

~~9. Werle, E», and Roden, P« Uber das vorkommen von kallikrein in den speicheldr sen und in mundspeichel. Biochem Z. 286: 213, 1036.~~

10. Ungar, G. and Parrot, J. L. Sur la presence de la callicreine dans la salive, et la possibilite de son intervention dans la transmission chemique de lHnflux nerveux. Compt. Rend. Soc. Biol. 122: 1052, 1936.

~~II. Hilton, S. M. and Lewis, G. P. The cause of the vasodilatation accompanying activity in the submandibular salivary gland. J. Physiol. 128: 235, 1955.~~

~~12. _, The mechanism of the functional hyperaemia in the submandibular salivary gland. J. Physiol. 129: 253, 1955. (~~

~~oi " " j.' ?C O~~

~~< -ioo~~

~~c* 30~~

~~13. , The relationship between glandular activity, bradykinin formation and functional vasodilatation in the submandibular salivary gland. J. Physiol. 134: 471, 1956.~~

~~14. Bhoola, K. D. , Morley, J. , and Schachter, M, Vasodilatation in the submaxillary gland of the cat. J. Physiol. 165: 36P, 1963.~~

~~15. Morley, J., Schachter, M., and Smaje, L. H. Vasodilatation in the submaxillary gland of the rabbit. J. Physiol. 187: 595, 1966.~~

~~16. Bhoola, K. D. , Morley, J., Schachter, M. and Smaje, L. H„ Vasodilatation in the submaxillary gland of the cat. J. Physiol. 179: 172, 1965.~~

~~17. Schachter, M. and Beilenson, S. Kallikrein and vasodilatation in the submaxillary gland. Gastroenterology 52: 401, 1967.~~

~~18. Skinner, N. S. and Webster, M. E. Submaxillary gland blood flow: the role of kinin and B adrenergic receptors. Fedn. Proc. 27: 76, 1968.~~

~~19. Beilenson, S„, Schachter, M., and Smaje, L„ H. Secretion of kallikrein and its role in vasodilatation in the submaxillary gland. J. Physiol. 199: 303, 1968.~~

~~20. Gautvik, K. M., Nustad, K. and Vystyd, J. Keninogenase activity in the stimulated submandibular salivary gland in the cat. Scand. J. Clin. Lab. Invest. 24: Suppl. 107: 101 +, 1969.~~

~~21. Gautvik, K. Studies on kinin formation in functional vasodilatation of the submandibular salivary gland in cats. Acta. Physiol. Scand. 79: 174, 1970.~~

~~22. Spock, A., Heick, H. M. C. , Cress, H. and Logan, W. S. Abnormal serum factor in patients with cystic fibrosis of the pancreas. Pediat. Res. 1: 173, 1967.~~

~~23. Bowman, B. H. , Lockhart, L. H. and McCombs, M. L. Oyster ciliary inhibition by cystic fibrosis factor. Science 164: 325, 1969.~~

~~24. Haverback, B. J., Dyce, B. J., Wong, T. and Swanson, V. L. Increase in trypsin binding of serum in cystic fibrosis. Clin. Res. 17: 148, 1969.~~

~~25. Lieberman, J. and Littenberg, G. D. Increased kallikrein content of saliva from patients with cystic fibrosis of the pancreas. Pediat. Res0 3: 571, 1969.~~

26. Schachter, M. Some properties of kallidin, bradykinin and wasp venom kinin. in Symposium. Polypeptides which affect smooth muscles and blood vessels, p. 235 ed. M, Schachter, London, Pergamon, I960.

~~27. Mandel, I. D. , Kutscher, A., Denning, C. R., Thompson, R. H. and Zegarelli, E. V. Salivary studies in cystic fibrosis. Amer. J. Dis. Child. 113: 431, 1967.~~

28. Mandel, I. D., Eriv, A., Kutscher, A., Denning, C. R., Thompson, R. H. , Kessler, W. , and Zegarelli, E. Calcium and phosphorous levels in submaxillary saliva. Changes in cystic fibrosis and asthma. Clin, Pediat. 8: 161, 1969.

~~29. Chernick, W. S., and Barbero, G. J. Studies on submaxillary saliva in cystic fibrosis. J. Pediat. 59: 890, 1961.~~

~~30. Chauncey, H. H., Levine, D. M. , Kass, G. , Shwachman, H., Henriques, B. L. and Kulczyeki, L. L. Composition of human saliva. Arch. Oral Biol. 7: 707, 1962.~~

~~31. Marmar, J., Barbero, G. J. and Sibinga, M. S. The pattern of parotid gland secretion in cystic fibrosis of the pancreas. Gastroenterology 50: 551, 1966.~~

~~32. Enrich, H. M., Stoll, E. , Friolet, B. , et al. Sweat composition in relation to rate of sweating in patients with cystic fibrosis of the pancreas. Pediat. Res. 2: 464, 1968.~~

~~33. Chernick, W. S. , and Barbero, G. J. Composition of tracheo¬ bronchial secretions in cystic fibrosis of the pancreas and bronchiectasis. Pediatrics 24: 739, 1959.~~

~~34. Kopito, L. and Shwachman, H. Mineral composition of meconium. J. Pediat. 68: 313, 1966.~~

~~35. Kopito, L. and Shwachman, H. Spectroscopic analysis of tissues from patients with cystic fibrosis and controls. Nature 202: 501, 1964. o 32~~

~~36. Ryan, J. W. , Roblero, J. and Stewart, J. M. Inactivation of bradykinin in the pulmonary circulation. Biochem. J. 110: 795, 1968.~~

~~37. Brocklehur st, W. E. , and Zeitlin, I. J. Determination of plasma kinin and kininogen levels in man. J. Physiol. 191: 417, 1967.~~

~~38. Dreisbach, R. H. Secretion of calcium by the rat submandibular gland. Amer. J. Physiol. 196: 645, 1959.~~

~~39. Nielsen, S. P. and Petersen, O. H. Excretion of magnesium, calcium, and inorganic phosphate by the cat submandibular gland. Pflueger. Arch. 318: 63, 1970.~~

~~40. Young, J. A. and Schogel, E. Micropuncture investigation of sodium and potassium excretion in rat submaxillary saliva. Pfluegers, Arch. ges. Physiol. 291: 85, 1966.~~

~~41. Petersen, O. H. and Paulsen, J. H. Excretion of sodium and potassium in cat submandibular saliva. Acta. Physiol. Scand. 70: 158, 1967.~~

~~42. Kellermeyer, R. W, and Graham, R. C. Kinins. Possible physiologic and pathologic roles in man. NEJM 279*. 14, 15, 16, 754, 802, 859 1968.~~

~~43. Schachter, M. Kallikreins and kinins. Physiol. Rev. 49: 509, 1969.~~

~~44. Mangos, J. A., McSherry, N. R. and Benke, P. J. A sodium transport inhibitory factor in the saliva of patients with cystic fibrosis of the pancreas. Pediat. Res. 1: 436, 1967.~~

~~45. Gibson, L. E., Matthews, W. J. Jr., and Minihan, P. T. Hyperpermeable mucus in cystic fibrosis. Lancet, 25 Jul 1970: 189.~~

~~46. Gugler, E. , Pallavicini, C. J. , Swederlow, H. and di Sant’Agnese, P. A. The role of calcium in submaxillary saliva of patients with cystic fibrosis. J. Pediat. 71: 585, 1967.~~

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~~YALE MEDICAL LIBRARY~~

~~Manuscript Theses~~

Unpublished theses submitted for the Master's and Doctor's degrees and deposited in the Yale Medical Library are to be used only with due regard to the rights of the authors. Bibliographical references may be noted, but passages must not be copied without permission of the authors, and without proper credit being given in subsequent written or published work.

~~This thesis by has been used by the following persons, whose signatures attest their acceptance of the above restrictions.~~

~~NAME AND ADDRESS DATE~~