Cyclic Nucleotide Levels in the Mammary Glands of Rats During Late Pregnancy and Early Lactation Fredrick William George Iowa State University

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1975 Cyclic nucleotide levels in the mammary glands of rats during late pregnancy and early lactation Fredrick William George Iowa State University

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Xerox University IMicrofilms 300 North Zeeb Road Ann Arbor, Michigan 48106 I 76-1838 GEORGE, Fredrick William, 1946- CYCLIC NUCLEOTIDE LEVELS IN THE MAMMARY GLANDS OF RATS DURING LATE PREGNANCY AND EARLY LACTATION. Iowa State University, Ph.D., 1975 Chemistry, biological

Xerox University l\Alcrofilm8, Ann Arbor, Michigan 4eioe

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. Cyclic nucleotide levels in the mammary glands of rats

during late pregnancy and early lactation

Fredrick William George

A Dissertation Submitted to the

Graduate Faculty in Partial Fulfillment of

The Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Department: Zoology Major: Zoology (Physiology)

Approved ;

Signature was redacted for privacy. in Charge of Ma; work

Signature was redacted for privacy. For the Major Department

Signature was redacted for privacy. For (rhg g&aduate College

Iowa State University Ames, Iowa

1975 ii

TABLE OF CONTENTS

Page

INTRODUCTION 1

LITERATURE REVIEW 1

Prelactational Development 4

Lactational Development and Lactogenesis 7

Cyclic Nucleotides and Hormone Action 19

Cyclic Nucleotides in Mammary Tissue 30

METHODS 32

Treatment of Animals 32

Tissue Preparation 32

Nucleic Acid Determinations 33

Cyclic Nucleotide Analysis 34a

Effect of Oxytocin on Cyclic GMP Levels 34a

RESULTS 35

Nucleic Acid Analysis 35

Cyclic Nucleotide Analysis 40

Effect of Oxytocin 42

DISCUSSION 51

SUMMARY 57

LITERATURE CITED 58

APPENDIX A: NUCLEIC ACID AlfALYSIS 72

Removal of Lipids from Mammary Glands 72

Procedure for Extracting Total Nucleic Acid 73

Preparation of Stock Hydrolyzed DNA and RNA Solutions 74 iii

Page

Colorimetric Determination of DNA Content 75

Colorimetric Determination of RNA Content 76

APPENDIX B; CYCLIC NUCLEOTIDE ANALYSIS 81

Preparation of Tissue Samples for Cyclic Nucleotide Analysis 81

The Assay 81

Calculations 83

ACKNOWLEDGMENTS 84 1

INTRODUCTION

Growth and differentiation of the mammary gland are dependent on the actions of a number of hormones acting in con cert throughout pregnancy and lactation. Lactogenesis, in particular, is controlled by changes in the hormonal environ ment at the time of parturition and is characterized by an in crease in ribonucleic acid (RNA) and the ribonucleic acid to deoxyribonucleic acid (RNA;DNA) ratio (Denamur 1974), lactose

(Shinde et al. 1965; Kuhn and Lowenstein 1967), a-lactalbumin

(Kuhn 1968), casein (Liu and Davis 1967) and fatty acid synthesis (Patton and Hood 1969). Also, the onset of lacta- tation is preceded by a sharp increase in the activities of many enzymes (Baldwin and Milligan 1966).

DNA synthesis has been reported by several workers to con tinue into the first few days of lactation (Griffith and

Turner 1961; Greenbaum and slater 1957; Kunford 1963; Traurig

1967) supporting the idea of a wave of cell proliferation in the mammary gland beginning at parturition and continuing through the first 3 days of lactation (Greenbaum and Slater

1957).

In spite of the fact that there is a great deal of infor mation available on the hormonal requirements for mammogenesis, lactogenesis and galactopoiesis, the cellular mechanisms of hor

monal stimulation involved are not clearly understood. Cyclic 2 adenosine 3',5'-monophosphate (cyclic AMP) has been implicated as a a second messenger in hormonal stimulation (Sutherland et al. 1965) and has been implicated in the modification of nuclear proteins resulting in changes in the rate of synthesis of multiple classes of RNA (Jost and Sahib 1971). Majumder and

Turkington (1971b) reported two protein kinases from mouse mammary cells, one of which was activated by cyclic AMP.

Sapag-Hagar and Greenbaum (1973) reported changes in rat mammary gland cyclic AMP, phosphodiesterase and adenylate cyclase activities during pregnancy and lactation. Peak activities of adenylate cyclase and cyclic AMP were reported on the 20th day of pregnancy while activities were found to decline throughout lactation. Phosphodiesterase activities were low during pregnancy and high during lactation. Subse quently, Sapag-Hager et al. (1974) reported the inhibition of DNA, RNA and fatty acid synthesis in rat mammary organ cultures incubated in Medium 199 containing 10"^ M dibutyrl cyclic AMP.

Recently, Goldberg et al. {1973a,b) have presented evidence

for a reciprocal relationship betv/een cyclic AMP and cyclic

guanosine 3',5'-monophosphate (cyclic GMP) in a number of

experimental situations. Of particular interest were reports

that high cyclic AMP levels are capable of inhibiting cell

proliferation (Abe11 and Monahan 1973; Otten et al. 1971) and 3 that an increase in cyclic GMP was observed in mitogenic stimu lated proliferation of human blood lymphocytes (Hadden et al.

1972).

Since cyclic GMP levels have not been reported for rat mammary tissue, it was thought desirable to evaluate both cyclic AMP and cyclic GMP levels in relation to DNA and RNA levels in mammary glands of pregnant and lactating rats in hopes of attaching some significance to the levels of these cyclic nucleotides in relation to proliferation and protein synthetic capabilities of the gland. It was also thought important to determine if the cyclic nucleotides in the mammary gland were related in a reciprocal manner, according to the theory of Goldberg et al. (1973b). 4

LITERATURE REVIEW

Prelactational Development

The hormonal factors influencing mammogenesis have been extensively studied both in vivo and ^ vitro and have been the subject of many reviews (Lyons, Li and Johnson 1958;

Jacobsohn 1961; Cowie 1966; Turner 1970; Anderson 1974; Ceriani

1974; Porter 1974). The following is a brief review of the development of the functional lactating mammary gland.

Pregestational development

The mammary glands develop from the embryonic region known as the mammary band. Mammary buds develop from the Malpighian layer of the skin and differentiate into primary and secondary sprouts. In the female, the secondary sprouts form the basis of the functional duct system. Post partum growth, prior to puberty.- is primarily characterized by an increase in connective tissue and fat deposition. A period of isometric growth pre cedes a period of allometric development where mammary gland growth rate exceeds the rate of increase in body surface area.

This change from isometric to allometric growth occurs approxi mately at the time of weaning and well before the onset of puberty. Castration prevents allometric growth suggesting that ovarian steroids are important during this stage. After

puberty, further development characterized by branching and re 5 branching of the ductal network takes place with each recurring estrous cycle. Maximal virginal development of the mammary glands in rats is usually obtained by the fourth estrous cycle and further development requires the altered hormonal environ ment of pregnancy (Anderson 1974),

Mammoqenesis during pregnancy

With the onset of pregnancy the mammary gland of the rat undergoes a sequential transformation characterized by proliferation of the ductal network, differentiation of the epithelium to form the secretory cell clusters at the termina tions of the ducts, and milk synthesis. Proliferation of the mammary gland during pregnancy occurs primarily in the paren chymal portion of the gland and at the cost of the fat pad

(Harkness and Harkness, 1956; Paape and Sinha 1971).

Originally, cellular proliferation of the mammary gland

was thought to be complete during the first half of gestation

(Weatherford 1929; Turner and Schultze 1931). This was chal

lenged by Jeffers in 1935 and in more recent studies which

demonstrated that cellular proliferation continued throughout

pregnancy and into the first few days of lactation (Greenbaum

and Slater 1957; Griffith and 'Turner 1961; Munford 1963;

Traurig 1967). Greenbaum and Slater (1957) observed a doubling

of total mammary gland DNA between parturition and day 3 of

lactation, while Griffith and Turner (1961) reported that 6 greater than 40 per cent of the mammary gland development occurred during early lactation. Traurig (1967) found sig nificant incorporation of tritiated thymidine into mammary gland DNA on days 2 and 3 of lactation. These later studies support the idea that there is a wave of epithelial cell proliferation occurring in the mammary gland after parturition during early lactation.

Hormonal factors

The hormonal control of mammogenesis during pregnancy is complex and involves the interaction of many hormones.

The ovarian steroids were the first hormones to be studied in relation to mammary gland development. In rats and mice, estrogen stimulates ductal proliferation while progesterone, in the presence of small amounts of estrogen, is necessary for lobuloalveolar growth (Turner 1939; Moon et al. 1959).

Several investigators pointed out the importance of the hormones of the anterior pituitary gland in mammogenesis when they demonstrated that ovarian steriods failed to have a mam- mogenic influence in hypophysectomized rats and mice (Nelson and Tobin 1936; Reece, Turner and Hill 1936; Selye and Collip

1936). Nathanson et al. (1339) and Reece and Leonard (1941) subsequently demonstrated mammary gland duct growth in hypo physectomized rats injected with estrogen and "crude" pitui tary extracts. 7

Extensive work by Lyons, Li and Johnson (1958) helped to emphasize the broad base of hormonal support the mammary gland receives in its development. In triply operated (hypophysectomized-adrenalectomized-ovariectomized) rats they demonstrated that estrogen, growth hormone (GH) and adrenal steroids were necessary for duct growth and that lobuloalveolar development was dependent upon the addition of progesterone and prolactin to the aforementioned complex.

The placenta may play an important role in mammogenesis during the last half of gestation in the rat. Matthies (1967) demonstrated the presence of a substance(s) in the tropho blastic elements of the midgestational rat placenta possessing luteotropic and mammotropic properties which he called rat chorionic mammotropin (RCM), Furthermore, Cohen and Gala

(1969) demonstrated luteotropic activity, which they at tributed to RCM, in the serum of pregnant rats on the 12th day of gestation, but they determined that the mammotropic

activity of this serum was modest, at best, and was incapable

of inducing secretory activity.

Lactational Development and Lactogenesis

The initiation of lactation (lactogenesis) is character

ized by a sudden increase in the secretory activity of the

epithelial cells brought forth by the complex interaction of 8

hormonal and neural stimulation occurring around the time of

parturition (Cowie 1969; Denamur 1971; Kuhn 1971; Tucker 1974).

Hormonal factors

The current concept of a triggering mechanism for lacto-

genesis involves removal of the progesterone block at par

turition and the positive stimulus of the "lactogenic complex"

from the anterior pituitary (cowie 1969).

In the rat, hypophysectomy causes the cessation of milk

synthesis (Selye et al. 1933) and reduces mammary cell numbers,

DNA, RNA and several enzymes essential for milk synthesis

(Baldwin and Martin 1968). Hormonal replacement studies in

hypophysectomized rats demonstrated that prolactin and adre

nocorticotropic hormone (ACTH) or adrenal corticoids are re

quired for initiation and for maintenance of lactation (Nelson

et al. 1943; Bintarningsih et al. 1957, 1958; Cowie 1957).

Lyons, Li and Johnson (1958), working with the triply operated

rat found that prolactin (mammotropin) and adrenal corticoid

were the hormones necessary for lactational development, al

though somatotropin and thyroxine enhanced the action of the

former two hormones.

Barrena (1975) has reported that adrenalectomy in rats on

day 18 of pregnancy caused a significant reduction in mammary

. cell size and RNA:DNA ratios when compared to controls sacri

ficed on the 22nd day of pregnancy. Davis and Liu (1969) have 9 demonstrated that adrenalectomy on the 14th day of pregnancy inhibits secretion of casein-like material in the mammary glands of rats in which lactation was experimentally induced by ovariectomy.

Biologically active corticosteriods may be present at too low a level during pregnancy, due to the presence of high circulating levels of corticosteriod binding globulin (CBG) to allow for milk secretion. Gala and Westphal (1965) noted a precipitous drop in CBG activity of rat serum from day 20 of pregnancy to day 3 of lactation and postulated that this decrease in CBG resulted in an increase in free corticosteriod which is important for initiation and maintenance of lacta

tion.

It is thought that estrogen and progesterone render the mammary gland secretory cells refractory to stimulation by prolactin and adrenal corticoids during pregnancy.

The combination of estrogen and progesterone during preg nancy has been shown to inhibit milk secretion (Meites

and Sgouris 1953)^ and lactation has been induced in pregnant

rats by ovariectomy (Liu and Davis 196?)=

Kuhn (1969a) has reported that progesterone administra

tion completely inhibits both experimentally-induced and

normal lactogenesis. At parturition there is a drop in the

circulating progesterone levels and a shift in the estrogens

progesterone ratio. Decline in progesterone levels at 10 parturition are probably due, in part, to increased conversion of this steriod to 20 a-hydroxy-4-pregnen-3-one (20 a-OH-P).

Fajer and Barraclough (1967), Hashimoto et al. (1968),

Wiest et al. (1968) and Labhsetwar and Watson (1974) have reported a drop in progesterone and an increase in serum

20 a-OH-P levels prior to parturition in rats. Wiest and

Forbes (1964) and Talwalker et al. (1961) have reported the

20 a-reduced compound to have much less hormonal activity than progesterone,and Kuhn (1969b) has reported that this compound does not inhibit laetogenesis.

Yoshinaga et al.. (1969) and Shaikh (1971) have demon

strated an increase in estrogen prior to parturition in the

rat. The initiation of milk secretion at term has been at

tributed to this shift in ovarian steriods in favor of estro

gen which stimulates prolactin release from the anterior

pituitary (Meites and Sgouris 1954).

Progesterone has also been shown to partially inhibit the

stimulatory effects of estrogen on prolactin secretion in rats

(Yokoyama et al. 1969; Chen and Meites 1970). Liu and Davis

(1967) and Kuhn (1969a) have demonstrated that ovariectomy or

other means of removing progesterone from the circulation of

mid-or late pregnant rats will induce lactation.

It has also been demonstrated that exogenous prostag

landin (PGFgg) induces laetogenesis and abortion in 11 pregnant rats (Deis 1971). Labhsetwar and Watson (1974) demonstrated, however, that the progesterone decline prior to parturition in the rat preceded, and was not associated with, the rise in PGF at parturition.

Oxytocin and the neuroendocrine factors involved during parturition and suckling may also be of importance in the initiation and maintenance of milk secretion. Folley and

Knaggs (1965) demonstrated the release of considerable quanti ties of oxytocin during parturition in the goat. Bryant,

Greenwood and Linzell (1968) reported that the intravenous in jection of oxytocin to goats causes an abrupt increase in plas ma prolactin. Also, the suckling stimulus of the newborn elicits a rapid release of prolactin, adrenocorticotropic hor mone (ÀCTH) and oxytocin from the pituitary gland (Meites

1959).

The hormonal control of lactogenesis can be summarized

in the following manner. During pregnancy, estrogen and pro

gesterone directly inhibit the secretory activity of the

mammary epithelium. In addition, progesterone interferes with

the estrogen stimulated release of prolactin from the

anterior pituitary. Biologically active corticosteriods are

kept low during pregnancy by relatively high levels of corti-

costeriod binding globulin. At parturition the decrease in

corticosteriod binding activity could account for an increase

in biologically active corticoids. Also, decreased progester 12 one secretion and increased estrogen secretion are responsible for an important shift in the estrogen:progesterone ratio.

Prolactin release from the anterior pituitary and removal of the "secretory block" on mammary epithelium could be related to a more favorable estrogen:progesterone ratio and to stimu

lation by high levels of oxytocin at parturition and in

response to suckling.

Biochemical changes during lactogenesis

Rivera (1974) emphasized the need for a more precise

definition of lactogenesis than the use of appearance of

milk in the alveoli as the end point for assessing lacto

genesis. This is of particular importance in in vitro

studies since the appearance of milk secretions is seldom

evident. Biochemical markers such as casein and lactose

serve as a more precise qualitative and quantitative assess

ment of response.

The^ onset of milk secretion is accompanied by an eleva

tion in mairanary RNA (Denamur 1969, 1974) and DNA (Baldwin 1969), ^

casein (Liu and Davis 1967), lactose (Yokoyama, et al. 1969;

Kuhn 1968; 1971) and fatty acid synthesis (Popjak et al.

1949; Fatton and Hood 1369). Also, the levels of several

constituitive enzymes of the pentose cycle, glycolysis and

citric acid cycle increase during late pregnancy (Baldwin

and Yang 1974; Baldwin 1969). These changes reflect changes 13 in the synthetic capacity and energy requirements of the active secretory cell.

Turkington et al. (1973) have suggested that cell pro liferation is a necessary prelude to the development of cell differentiation and the attainmeht of secretory function in cultured mammary cells. Stockdale and Topper (1966) demon strated that DNA synthesis (cell division) was necessary for the initiation of differentiation as assessed by casein synthesis in mouse mammary gland cultures. This would appear to be satisfied vivo in the rat since DNA synthesis occurs throughout the period of lactogenesis from late pregnancy to early lactation (Baldwin 1969).

The rate of increase in enzyme activities (phosphogluco-

mutase, citrate cleavage enzyme, uridine diphosphate (UD?)

galactose-4-epimerase and glucose-6-phosphate dehydrogenase)

has been shown by Baldwin and Milligan (1966) to be 3- to

4-fold per mg DNA. This illustrates an increase in the

enzymatic capabilities of the individual secretory cells

rather than simply an increase in enzyme activity of the

mammary gland due to an increase in the number of cells.

Recently Oka and Perry (1974) demonstrated that insulin,

insulin and prolactin, and insulin, prolactin, and hydrocorti

sone produce 100%, 200% and 250% elevations respectively in

the activity of glueose-C-phosphate dehydrogenase activity 14 of mouse mammary epithelial cells in culture. It was also shown that glucose-6-phosphate dehydrogenase activity de clined after two days in insulin and insulin + prolactin stimulated cultures, but that enzyme activity was main tained at high levels for 4 days in the cultures containing hydrocortisone.

Popjak et al. (1949) reported a 10-fold increase in the synthesis of short chain fatty acids from acetate at parturi tion in rats. These findings are consistent with an in crease in the respiratory quotient (R.Q.) of lactating rat mammary slices ^ vitro reported by Folley and French (1949).

Gumaa et al. (1973) reported an increase in the activities of several mammary enzymes concerned with fatty acid synthesis

(acetyl CoA carboxylase and fatty acid synthetase) and the

provision of acetyl CoA units (citrate cleavage enzyme) for

fatty acid synthesis over the period of parturition in the

rat. Also increases in the activities of glucose-6-phosphate

dehydrogenase and "malic" enzyme, both of which serve as a

source of reducing agents (NADPH) for fatty acid synthesis,

were reported over this same period. The uptake of plasma

triglycerides at parturition is reflected in a 100=fold in

crease in the activity of lipoprotein lipase (McBride and

Korn 1963).

Lactose and the lactose synthetase enzyme complex have

been studied extensively in relation to lactogenesis and the 15 hormonal factors initiating milk secretion.

Lactose synthetase is rate limiting in the synthesis of lactose in the mammary gland (Kuhn 1971, Baldwin and Yang

1974). In relation to its rate limiting effects, Kuhn (1969b) noted only small changes in the activities of hexokinase, phosphoglucomutase, UDP glucose pyrophosphorylase and UDP- glucose-4-epimerase (enzymes in the lactose pathway) but a

15-fold increase in the acti\ity of lactose synthetase in mammary glands of rats during the last two days of pregnancy.

Kuhn (1968) and Yokoyama et al. (1969) detected lactose synthe tase activity and lactose, respectively, in rat mammary glands the day before parturition. Artificial removal of progesterone stimulus by bilateral ovariectomy was also shown to initiate lactose synthesis.

The lactose synthetase enzyme catalyzes the formation of lactose from UDP-galactose and glucose and is a complex enzyme made up of two components: the catalytic unit, galactosyl- transferase (A-protein) and a specifier protein, a-lactalbumin

(B protein).

Yokoyama et al. (1969) reported that in rats lactose could

first be detected on the 20th day of pregnancy and was fol

lowed by an abrupt increase on the 21st day (the day before

parturition). They were also able to demonstrate an increase

in the lactose content of mammary glands in response to 16 ovariectomy on the 18th day of pregnancy.

Turkington et al. (1968) demonstrated that prolactin stimulated the synthesis of both A and B proteins in mouse mammary cultures pretreated with insulin and hydrocortisone.

They also measured the levels of these two proteins in mammary expiants from mice at different stages of pregnancy. The level of the A protein was found to increase to a maximum at the end of pregnancy while the B protein remained low throughout pregnancy, but rose dramatically at the initiation of lactation. They postulated that the asynchronous expression of A and B proteins represented a possible site for hormonal control of a unique biosynthetic pathway. Turkington and Hill

(1969) extended these studies demonstrating that progesterone repressed a-lactalbumin synthesis.

In contrast to studies in mouse mammary expiants, Kuhn

(1968) noted a marked increase in galactosyl transferase co

incident with lactogenesis in the rat.

Molecular mechanisms

The molecular mechanisms involved in the hormonal stimu

lation of specific milk proteins in mouse mammary expiants have

been the subject of intensive work by Turkington et ai.

(1973). A characteristic sequence of hormonal stimulation

including 1) insulin induction of stem cell division

2) hydrocortisone stimulated differentiation of daughter cells 17 followed by 3) prolactin (and insulin) stimulation is necessary for the induction of the milk proteins casein and lactose synthetase in cultured mammary cells.

In properly primed mammary cultures the addition of prolactin stimulates the appearance of all classes of RNA

(Turkington et al. 1973) followed by the appearance of casein and both A and B proteins of lactose synthetase. Thus, the induction of specific milk proteins by prolactin is preceded by the activation of transcription of multiple classes of

RNA.

Gene activation and transcription are probably regulated by DNA-associate histone and non-histone acidic protein (HHP)

(Monahan and Hall 1974). Studies of mammary epithelial cell

histones have demonstrated that these proteins lack the

specificity for regulating the transcription of the vast number

of genes on a DNA molecule. The non-histone acidic proteins

(NHP), in contrast to the histones, are highly species-and tissue-specific (Gilmour and Paul 1969; Kleinsmith et al. 1970).

Turkington et al. (1973) have reported differences in the

electrophoretic patterns of NHP isolated from mammary glands

of virgin and lactating mice and that DNA and NKP synthesis

in mammary expiants is stimulated by insulin, but not hydro

cortisone and/or prolactin. Therefore, accumulation of these

proteins is stimulated by factors affecting cell proliferation. 18 but not by the hormones which stimulate milk protein synthesis in differentiated alveolar cells in culture. These results emphasize the role of mitosis in mammary cell differentiation and Turkington (1971) has demonstrated that DNA replication and cell division are required for the derepression of the genes

for milk protein synthesis ^ vitro.

Turkington and Riddle (1969) have demonstrated the in- 32 corporation of P from ATP into histones and non-histone

nuclear protein and that phosphorylation of these nuclear pro

teins was temporally associated with activation of transcrip

tion by insulin and prolactin. Subsequently, Turkington and

Riddle (1970) and Kadohama and Turkington (1973) have demon

strated that the nuclear proteins phosphorylated are primarily

the ^2^3 ^2" histones and all species of acidic nuclear

proteins.

Majumder and Turkington (1971a) isolated two protein

kinases (I and II) in cytosol of mammary cells which demon

strated a high degree of specificity for histones. The

activity of protein kinase II was markedly stimulated by

cyclic AMP. It was reported that cyclic AMP interacts

with protein kinase II causing the release of a cyclic AMP-

binding subunit and the active catalytic subunit protein

kinase I. Protein kinase and cyclic AMP-binding protein activities have 19 been demonstrated to increase approximately 7- to 9-fold from virgin animals to day 20 of pregnancy followed by an un explained precipitous drop at the time of parturition

(Majumder and Turkington 1971a). In addition, these same workers reported that the cyclic AMP-dependent protein kinase could be induced in insulin-stimulated mitotic cells solely by prolactin. Furthermore, neither prolactin nor insulin stimulated adenylate cyclase activity in isolated mammary cell membrane.

It was proposed that the possible mechanism for regu lation (of phosphorylation of nuclear proteins) was controlled by the rate of synthesis of a cyclic AMP-dependent enzyme rather than the level of the cyclic nucleotide.

Cyclic Nucleotides and Hormone Action

Cyclic nucleotides apparently regulate many different physiological phenomena and have been implicated as inter mediates in hormonal stimulation (Sutherland 1972; Robison^,, et al. 1971; Goldberg et al. 1973b; Major and Kilpatrick 1972;

Harâman 1974).

The concentration of cyclic adenosine 3'-5'-monophosphate

(cyclic AMP) and cyclic guanosine 3',5'-monophosphate (cyclic

GMP) represent only a small fraction of the total adenine or guanine nucleotide content of tissue. Cyclic AMP concentra tions are reported to be in the range of 0.1-1.0 y mol/kg wet 20 tissue (Hardman 1974) and cyclic GMP concentrations are usually 1/10 to 1/100 the levels of cyclic AMP (Goldberg et al.

1973a).

Cyclic AMP is synthesized from adenosine triphosphate

(ATP) by membrane bound adenylate cyclase (AC). Magnesium

(Mg^^) or manganese (Mn^^) is required for enzyme activity

(Hardman 1974). Degradation of the cyclic nucleotide is controlled by hydrolysis of the 3'bond by a phosphodiesterase to form 5'-adenosine monophosphate (5'-AMP).

Cyclic GMP is synthesized from guanosine triphosphate

(GTP) by the enzyme guanylate cyclase (GC). The reaction appears to be analogous to the formation of cyclic AMP from adenosine triphosphate (ATP), however, several differences have been found with respect to subcellular distribution and substrate and cation requirements (Goldberg et al. 1973a).

Unlike adenylate cyclase, which has been reported to

occur predominantly in the plasma membrane fractions of tissue

homogenates (Perkins 1973), the major portion of guanylate

cyclase appears to reside in the soluble fraction after

homogenization of most tissues (White and Aurbach 1969;

Hardman and Sutherland 1969), The exact location is, how

ever, less certain than the location of adenylate cyclase

since, even in tissue homogenates where the majority of GC

activity appears to be cytoplasmic.- there is significant 21

(10-20%) activity associated with the membrane fraction.

Also, in some tissues (i.e. small intestine) the majority

of activity is found in the particulate fraction, whereas,

other tissues (i.e. heart) have intermediate distribution

between cytoplasm and membrane fractions (Goldberg et al.

1973a). One interpretation might be that the intracellular

distribution of guanylate cyclase is different in different

tissues; another explanation is that possibly the guanylate

cyclase becomes dissociated to a greater or lesser extent

during the homogenization procedure. Goldberg et al. (1973a)

feel: that the cytoplasmic location of guanylate cyclase in

certain tissues may be more apparent than real and may be due

to the fractionation procedures of in vitro experiments.

The requirement of guanylate cyclase activity for Mn^^

is another property which appears to distinguish it from ++ adenylate cyclase which can apparently utilize either Mg or

I* j Mn equally well. The guanosine triphosphate (GTP) to Mn

ratio may be an important factor in controlling guanylate

cyclase activity, since Hardman and Sutherland (1969) demon

strated that increasing GTP concentrations in the presence of

fixed,- low concentrations of inhibited enzyme activity.

Another interesting difference between the two cyclases

is the effect of calcium (Ca^^^ on the activity of the enzymes.

In most cases, the activity of adenylate cyclase is inhibited 22 by the presence of Ca**. Blrnbaumer et al. (1970) reported that hormonal stimulation of adenylate cyclase from fat cell ghosts is enhanced in the presence of a calciim chelator

(E6TA) with the one exception: that stimulation by ACTH was markedly inhibited by EGTA. In contrast Ca^* seems to stimulate guanylate cyclase activity. Hardman et al. (1972)

have demonstrated that Ca could enhance guanylate cyclase

activity several fold when present with Mn^^ and adequate 6TP.

These findings suggest that the ion composition of the cells

is an important factor in the regulation of the activity of

the cyclases.

Cyclic GMP is degraded in the cell by phosphodiesterase

hydrolysis of the 3' bond to form 5'-GMP similar to the de

gradation of cyclic AMP. Early investigation (Drummond and

Perrott-Yee 1961) seemed to indicate the presence of a

common phosphodiesterase, but more recent studies seem to

indicate the presence of specific phosphodiesterase enzymes

(Thompson and Appleman 1971; Kakiuchi et al. 1971).

The second messenger hypothesis

Sutherland, 0ye and Butcher (1965) introduced the second

messenger theory to explain the actions of a variety of amine

and polypeptide hormones. In this model the "first messenger"

or hormone interacts with its specific receptor on the cell

membrane which closely associated with the membrane bound 23 cyclase (adenylate cyclase). As a result of hormone-receptor interaction adenylate cyclase activity is either stimulated or suppressed with the respective increase or decrease in the

"second messenter", cyclic AMP. According to this concept the first messenger (hormone) carries the information to the cell and the second messenger (cyclic AMP) transfers the in formation internally to obtain the appropriate intracellular response.

Cyclic AMP is thought to act by activating protein kinases (Walsh st al. 1968), which subsequently phosphorylate a number of diverse proteins within the cell. Langan (1973) has reviewed the role of protein kinases and has made the following conclusions: 1) that protein kinases seem to be

involved in each of the well documented cases of cyclic AMP action and 2) that one cyclic AMP protein kinase, in spite of existing in multiple forms, is capable of mediating a

variety of hormone actions.

It is therefore probable that the response of a particu

lar cell to a rise in cyclic AMP is essentially determined by

the number and types of protein kinase substrates present.

The substrates are in turn regulated by the genetic expression

in the cell.

There is evidence for the presence of cyclic GMP-dependent

protein kinase in several systems although the substrate 24 specificities for this kinase are less clear than those for the cylic AMP-dependent kinase (Goldberg et al. 1973a). Before the natural concentration of cyclic GMP was known, Rail and Suther land (1962) demonstrated that cyclic GMP was 1/500 as potent as cyclic AMP in activating dog liver glycogen phosphorylase.

Schlender et al. (1969) demonstrated that, while both cyclic AMP and cyclic GMP were capable of stimulating rabbit

muscle glycogen synthetase-I kinase (cyclic AMP-dependent

protein kinase), the association constant (K^) for the two

cyclic nucleotides were 0.07 and 9»9 mM,respectively, Majumder

and Turkington (1971b) reported that interference with cyclic

AMP binding to mouse mammary gland protein kinase by cyclic GMP

occurs only at relatively high concentrations of the latter.

Recently, however, Goldberg et al. (1973a) demonstrated a 20-30%

inhibition of cyclic AMP binding to skeletal muscle protein

kinase by 10 to 10 M cyclic GMP. This appears to be a

significant amount of inhibition with cyclic GMP close to the

physiological range.

Kuo and Greengard (1969, 1970a) were the first to report

the occurrence of a protein kinase which seemed to be more

sensitive to cyclic GMP than to cyclic AMP stimulation. Pro

tein kinases were purified from lobster tail muscle showing

specificity for cyclic AMP and cyclic GMP. The protein kinase

more sensitive to activation by cyclic GMP (cyclic GMP= 25 dependent protein kinase) exhibited of 0.08 uM for cyclic

GMP and a value 50-fold higher for cyclic AMP. Interesting

ly, a protein factor isolated by Appleman et al. (1966) from mammalian and arthropod tissue and believed to be similar to the protein inhibitory subunit of cyclic AMP-dependent

protein kinase, was found by Kuo and Greengard (1971) to

stimulate cyclic GMP dependent protein kinase from lobster

muscle.

It is not, as yet, clear what the substrate or substrates

are for the cyclic GMP-dependent protein kinase, but Kuo and

Greengard (1970b) have compared the effects of cyclic AMP-

and cyclic GMP-dependent protein kinases on the phosphoryla

tion of histones. Although they found that both cyclic AMP

and cyclic GMP were able to utilize histones as substrate-

there were marked differences in the phosphorylation patterns

catalyzed by the cyclic nucleotide-dependent protein kinases.

They concluded that there were probably separate and inde

pendent roles for cyclic AMP and cyclic GMP and their re

spective protein kinases.

Recently, Johnson and Madden (1975) have reported that

cyclic GMP stimulates the incorporation of phosphate into

specific nuclear acidic proteins of horse peripheral blood

lymphocytes. Furthermore they determined that prostaglandin

(PGE^, which elevates cyclic AMP in lymphocytes) inhibited 26 the phosphorylation of acidic non-histone nuclear proteins.

The role of guanosine 5•-triphosphate (GTP) as a possible

activator of the adenylate cyclase was investigated by Leray

et al. (1972). Assay of adenylate cyclase activity in rat

liver plasma membranes revealed that GTP was capable of en

hancing the response of the enzyme to stimulation by both

glucagon and epinephrine. Siegel and Cuatrecasas (1974)

studied the effect of Mg and GTP on the regulation of fat

cell adenylate cyclase. They found that in the presence of 4*4* Mg , GTP is no longer required but that it enhances the

response to epinephrine. GTP was found to lower the appa

rent affinity of adenylate cyclase for ATP, while simultaneous

ly increasing the velocity of the catalytic reaction. It was

postulated that hormonal responsiveness is controlled by

varying substrate (ATP, GTP and Mg"^"^) concentrations.

Dualism theory

The "dualism theory" was proposed by Goldberg et al.

(1973a,b) to explain the reciprocal relationship between

cyclic AMP and cyclic GMP that had been reported by several

investigators. According to this theory, cyclic GMP produces

a cellular response opposite to or antagonistic to those that

occur when the concentration of cyclic AMP is elevated.

George et al. (1970) reported that acetylcholine (Ach)

perfused rat hearts produced an elevation of myocardial cyclic 27

GMP coincident with the first signs of cholinergically-induced depression of cardiac function. They found that cyclic GMP levels were elevated 280% 10 sec after Ach administration, then leveled at 150% above control values, 30 sec after ad ministration. Cyclic AMP was reported 40% lower, 20 sec after cholinergic treatment and returned to control levels by

30 seconds. Pretreatment of the hearts with atropine, a

muscarinic blocking agent, prevented depression of cardiac

function and elevation of cyclic GMP in response to Ach. Iso

proterenol, a strong adrenergic agent depressed cyclic GMP,

stimulated cyclic AMP accumulation and increased rate and

force of contraction.

Kuo and Kuo (1973) reported that isoproterenol increased

pulmonary cyclic AMP levels 3-fold, while Ach elevated cyclic

GMP levels 3-fold. The effects of isoproterenol or acetyl

choline were blocked by propranolol or atropine, respectively.

Oxytocin and serotonin have been reported to elevate

cyclic GMP levels in uterine smooth muscle with no signifi

cant effect on cyclic AMP (Goldberg et ai. 1973b). This is

reported to be the first demonstration of an increase in

tissue cyclic GMP associated with a peptide hormone and by a

neurohormone other than Ach.

Stone et al. (1975) reported opposing effects of cyclic

AMP and cyclic GMP on electrophysiologically identified pyra

midal tract neurons. Norepinephrine and cyclic AMP inhibited 28 the firing of these neurons while cyclic GMP and Ach excited the neurons. It was suggested that these two cyclic nucleo tides possibly function as reciprocal intracellular second messengers for norepinephrine and Ach, respectively.

In contrast to most reports which have associated cyclic

GMP with muscarinic cholinergic stimulation and cyclic AMP

with adrenergic stimulation, Ferrendelli et al. (1975) re

ported that norepinephrine (NE) elevates levels of both cyclic

AMP and cyclic GMP in incubated mouse cerebellar tissue.

Omission of Ca^^ from the incubation medium blocks the NE

stimulated accumulation of cyclic GMP but not cyclic AMP.

The role of Ca agrees well with previously cited reports

(Birnbaumer et al. 1970; and Hardman et al. 1972) which

demonstrated that Ca 4* "h was a stimulator of guanylate cyclase

and an inhibitor of adenylate cyclase.

Goldberg et al. (1974) demonstrated a reciprocal rela

tionship between cyclic nucleotides in bovine vein prepara

tions. They noted that prostaglandin (PGFgg) caused

contraction and an increase in the cyclic GMP to cyclic AM?

ratio. Conversely, prostaglandin E, stimulated relaxation and

a decrease in the cyclic GMP to cyclic AMP ratio.

Elevation of intracellular cyclic AMP inhibits cell pro

liferation in many systems (Abell and Monahan 1973). Otten

et al. (1971) have demonstrated that the intracellular level 29 of cyclic AMP was inversely related to the rate of DNA syn thesis in 13 separate cell lines. Furthermore, they found that in 3T3 cells transformed by simian virus 40 (SV40) there was a corresponding decrease in cyclic AMP levels associated with uncontrolled cell proliferation. Sheppard (1972) con firmed and extended these findings by demonstrating that prostaglandin E^tPGE^) increased intracellular cyclic AMP levels and inhibited growth in transformed 3T3 cells. Bannai and Sheppard (1974) have also reported an increase in cyclic

AMP levels of cultured 3T3 cells during normal contact in hibition.

Hadden et al. (1972) reported that phytohemaglutinin

(PHA) and concanavalin A stimulated proliferation of human blood lymphocytes, produced a 10- to 50-fold increase in cellu lar cyclic GMP concentrations and had no effect on cyclic AMP synthesis.

Voorhees et al. (1973) investigated cyclic nucleotide levels in the rapidly proliferating epithelium of psoriosis.

They found that gyclic AMP levels were relatively low and cyclic GMP levels relatively high in rapidly proliferating psoriatic lesions when compared with uninvolved epithelium. 30

Cyclic Nucleotides in Mammary Tissue

Other than the reports of Turkington and co-workers

(Turkington et al. 1973) on the role of cyclic AMP-dependent protein kinases in mouse mammary cultures, there exists few reported studies of the occurrence or role of cyclic nucleo tides in normal mammary tissue.

Bar (1973) has reported an epinephrine- and prostalgan- din-sensitive adenylate cyclase in rat and rabbit lactating mammary gland tissue. The enzyme activity from rat lactating mammary tissue was found to be low, i.e. below 2-3 p mbl/mg/ min in unstimulated tissue. Sodium fluoride (NaP) was observed to stimulate cyclase activity 2- to 4-fold. Epinephrine stim ulation was observed only after collagenase digestion of tissues and then only to a relatively small but highly sig nificant 70-80%. Oxytocin, vasopressin, glucagon, ACTH, thyrotropic hormone and PGE^ had no effect on enzyme activity^

The adenylate cyclase from rabbit mammary gland was responsive

to prostaglandins ^ and in addition to epinephrine.

Oxytocin, vassopressin, ACTH, thyrotropic hormone, glucagon,

parathyroid hormone and prolactin had no effect on enzyme

activityc

Sapag-Hagar and Greenbaum (1973) reported changes in

adenylate cyclase and phosphodiesterase activities and in

cyclic AMP levels of the rat mammary gland at various stages 31 of pregnancy and lactation. Cyclic AMP levels were found to increase during pregnancy to peak levels on day 20 of ap proximately 680 p mol/g on day 1 of lactation and continued to decline throughout lactation to approximately 200 p mol/g on day 16 of lactation. Adenylate cyclase activity was ob served to parallel the changes in cyclic AMP# while phospho diesterase rose during pregnancy and continued to rise through out lactation. In an attempt to more clearly define the role of cyclic AMP in the mammary gland, Sapag-Hagar et al.

(1974) subsequently investigated the activities of several enzymes from 10-day or 20-day pregnant rat mammary tissue.

Expiants were incubated in the presence or absence of dibutyrl cyclic AMP. Virtually every enzyme studied (including glucose-6-phosphate dehydrogenase, citrate cleavage enzyme and fatty acid synthetase) was depressed by the presence of cyclic

AMP in the incubation medium. Cyclic AMP also was noted to

inhibit the incorporation of radioactive precursors into

RNA, DNA and fatty acids. 32

METHODS

Treatment of Animals

Virgin, female rats of the Sprague-Dawley strain, weighing iaO-200 grams, obtained from Sasco, Omaha, Nebraska, were maintained on a commercial lab chow (Wayne) and water ad libitum. Light (8 a.m. to 10 p.m.) and temperature (25C) were controlled.

Six female rats were placed in breeding cages with one male. Vaginal smears of each rat were made in the morning, and the presence of sperm was considered as evidence of day one of pregnancy. Pregnant rats were transferred to smaller cages, one or two rats per cage and sacrificed on either day 14 (P14) or 20 (P20) of pregnancy or day 1 (LOI) or 3 (L03) of lactation (the day of parturition being desig

nated day 1 lactation).

Tissue Preparation

On the day of sacrifice the total body weight of each

animal was recorded. Small portions of mammary tissue from

the inguinal region of the animal were rapidly excised and

frozen in liquid nitrogen while the animal was under light

ether anesthesia. This tissue was used for analysis of cyclic

nucleotides. The rat was then sacrificed by decapitation.

The three remaining abdominal inguinal mammary glands were 33 excised in toto, weighed wet and transferred to chloroform: methanol for defatting as described in Appendix A. This tissue was used for nucleic acid analysis. Corrected body weights were calculated for each animal by subtracting the total uterine weight from total body weight.

Nucleic Acid Determinations

RNA and DNA were extracted from 25 mg samples of dried fat-free tissue (DFFT) in hot trichloroacetic acid (TCA) according to the method outlined by Schneider (1945) and modi fied by Ferreri (1971).

DNA and RNA content of the mammary glands were then both estimated by colorimetric procedures. DNA content was esti mated by the diphenylamine method of Burton (1956). RNA content was estimated by the orcinol procedure of Mejbaum

(1939)j as described by Schneider (1957).

The absorbance measured in the mammary gland extracts was compared to the absorbance measured in standards of known nucleic acid concentration. The standard solutions of hydrolyzed DNA and RNA were prepared according to the method out= lined by Webb and Levy (1955). The accuracy of the standards was established by colorimetrically determining the phosphorus content according to the method of Fiske and Subbarow (1925).

All colorimetric determinations were made on a Beckman

Model 24 Spectrophotometer. Tissues were extracted in dupli- 34a cate and each extract was assayed in duplicate for both RNA and DNA. The mean of four determinations was then calculated for each Scunple. Details of the extraction procedure and the colorimetric determinations of nucleic acids are outlined in

Appendix A.

Cyclic Nucleotide Analysis

Both cyclic AMP and cyclic GMP were analyzed using com mercially available radioimmunoassay kits (Schwarz/Mann,

Orangeburg, N.Y.). The radioimmunoassay of cyclic nucleo tides has been described by Steiner, Parker and Kipnis (1972) and Steiner et al. (1972). The protocol upon which the cyclic AMP and cyclic GMP radioimmunoassay kits are based follows procedures described by Steiner et al. (1972).

The preparation of tissue samples and the complete protocol are outlined in Appendix B.

For both assays, two extracts of each tissue sample were assayed in triplicate. The mean of six observations was calculated for each sample.

Effect of Oxytocin on Cyclic GMP Levels

Because oxytocin administration has been shown to ele

vate cGMP levels in the uterus (Goldberg et al. 1973b) a

preliminary experiment was performed to test the effect of 34b

exogenous oxytocin on the levels of cyclic GMP in the mammary gland.

Five, 10-day lactating rats that were nursing at least

6 pups were isolated from their litters for 10 hours. The dams were then anesthetized with sodium pentobarbitol (50 mg/kg body weight) thereby blocking the neuroendocrine re lease of oxytocin from the posterior pituitary in response to the suckling stimulus. A median incision was made in the skin above the abdominal mammary glands. The mammary glands were carefully dissected from the skin leaving teat connections intact. Six pups were transferred to the dam and allowed to suckle. A piece of mammary tissue (approxi mately 100 mg) was dissected from the dam after the pups had been allowed to suckle for 2 minutes and prior to the oxyto cin injection. This sample was considered the control sample or 0 (zero) time samples,- Oxytocin (2 u.s.P. units) was then injected intraperitoneally and mammary tissue samples (approximately 100 mg) were collected at 30, 90 and

300 seconds postinjection, frozen in liquid nitrogen and stored at -76 C until analysis by radioimmunoassay for cyclic

GMP. 35

RESULTS

Data are presented, for convenience, in two sections:

(1) nucleic acid analysis and (2) cyclic nucleotide analysis.

Nucleic Acid Analysis

Data for body weights, dried fat-free tissue (DFFT), total DNA, total RNA, DNA/lOOg body weight (index of cellu

lar ity) and RNA:DNA ratio (index of metabolic activity) are

presented in Table 1 and Figures 1 and 2. Significance levels

(Student's t-test) are presented in Figure 3 for comparison

between groups.

No significant differences in body weights were noted.

Maxranary gland DFFT increased significantly (P<0.01) from 223.8

mg on day 14 of pregnancy to 283.5 mg on day 20 of pregnancy.

A further significant increase was noted from the 20th day

of pregnancy to the 1st day of lactation (465.6 mg DFFT;

P<0.001). There was no significant difference in DFFT be

tween day 1 and day 3 (457.5 mg) of lactation.

The DNA levels of the mammary gland paralleled the

changes in DFFT content of the gland as can be seen in

Figure 1. There was a significant (p<0,005) increase in

total mg DNA/100 g body weight from day 14 (3.09) to day 20

(3.80) of pregnancy, followed by a significant (P<0.005) in- Table 1. Mammary gland nucleic acid content

No. Body, Total DNA Total (mg/100 g of DFFT^ RNAC RNA/DNA rats (9) (mg) of body (mgX wt)

P14 10 283 223.8+11. 8*^ 8.69+0. 43 11.60+0. 66 3.09+0. 16 1.33+0. 03

P20 10 294 283.5+18. 0 11.17+0. 44 18.01+1, JL5 3.80+0. 13 1.60+0. 07

LOI 10 273 465.6+15. 4 12.35+0. 51 31.03+1. 29 4.54+0. 16 2.52+0. 05

L03 10 290 457.5+19. 2 13.22+0. 52 33.77+1. 86 4.53+0. 10 2.55+0. 10

^Corrected for total uterine weight.

^Dry fat-free tissue.

^For three abdominal-inguinal mammary glands.

^Mean + S.E.M. 6.0 500 IIZI DNA laDFFT il m 5.0 400 o m§M o m mm o \ mm < u_ z u_ o 4.0 300 û o m o w z iiil _l < < I-o I- 200 o

PI 4 P20 LOI L03 Figure 1. Total mg DFFT and total mg DNA/100 g body weight in mammary glands of rats at days 14 ànd 20 of pregnancy (P14; P20) and days 1 and 3 of lactation (LOI; L03) 38

I o 2 H- CC.<

u < onZ

PiU PEG I 01 103

Figure 2. Rat mammary gland RNA:DNA ratios during pregnancy (P14; P20) and lactation (LOI; L03) 39

TOTAL MG DNA/lOO G BW

P20 0.005

LOI 0.001 0.005

L03 0.001 0.001 N.S. PI4 P20 LOI

DFFT

P20 0.01

LOI 0.00! 0.00!

L03 0.001 0.001 N.S. PI4 P20 LOI

RNA:DNA RATIO

P20| 0.005 1

LOI I0.001 I0.001 I I LOSjI 0.001 {0.001 } N.S. i

PI4 P20 LOI

Figure 3. Significance levels (Student's t-test) between all groups for nucleic acid analysis 40 crease from day 20 to day 1 of lactation (4.54). No sig nificant change was noted between day 1 and day 3 (4.53) of lactation.

The RNA:DNA ratio increased throughout pregnancy (P14=

1.33; P20=1.60) to the 1st day of lactation (2.52), with no change between the 1st and 3rd day of lactation (2.55). The significance levels between day 14 and day 20 of pregnancy and between day 20 of pregnancy and the first day of lacta tion were P<0.005 and P<0.001 respectively.

Cyclic Nucleotide Analysis

Differences in the number of rats per group in cyclic nucleotide analysis from the number of rats per group in nucleic acid analysis are accounted for by the discard of 2 outliers (lying >3 standard deviations from the mean) in group P20 and the presence of missing values in one rat from each of the other three groups.

The mean coefficient of variation (CV) between duplicate samples in the cyclic AMP assay was 54.35+27.47%, while the mean CV within samples was 5.64+1.31%. For the cyclic GMP assay the mean CV between duplicate samples was 23.95+4.27% and the within sample CV was 4.37+1.02%.

Data for p mol cyclic nucleotide per g wet mammary tissue as calculated directly from the assay are presented, along with values for total cyclic nucleotides in the mammary gland, in 41

Table 2. Graphie representation of the changes in cyclic nucleotides per g wet tissue over the assay period is in

Figure 4. Significance data are in Figure 8.

A significant (P<0.001 from P14; P<0.025 from LOI) peak in the concentration of cyclic AMP (390.5 p mol/g wet tissue) was noted on the 20th day of pregnancy. Values for p mol cAMP/g wet tissue for day 14 of pregnancy and days 1 and 3 of lactation were 143.33, 250.78 and 217.11, respectively. Days

14 of pregnancy and 3 of lactation were not significantly dif ferent, but day 1 of lactation was significantly different

(P<0.025) than day 14 of pregnancy. Values for p mol cyclic

GMP/g wet tissue were 5.36, 5.70, 7.97 and 8.07 for days 14 and 20 of pregnancy and days 1 and 3 of lactation, re

spectively. A significant (P<0.05) increase was noted between

days 20 of pregnancy and 1 of lactation.

Similar results were obtained when the cyclic nucleotides

were expressed per mg DNA (Table 3, Figures 5 and 8). A

significant increase in p mol cyclic AMP/mg DNA was noted

between days 14 (52.94) and 20 (132.31) of pregnancy. There

was a decline in levels from day 20 of pregnancy to day 1 of

lactation (100.53) which was not significant and a decline

from day 1 to day 3 (81.39) which was not significant. The

overall decrease in p mol cyclic AMP/mg DNA from day 20 of

pregnancy to day 3 of lactation was, however, significant

(P<0.05) Picomoles cyclic GMP/mg DNA were statistically the same 42 on days 14 (1.98) and 20 (1.88) of pregnancy. There was a significant increase between day 20 of pregnancy and day 1 of lactation (3.25 p mol cyclic GMP/mg DNA). The difference between the mean p mol cyclic GMP/mg DNA on day 1 of lactation and day 3 of lactation (2.84) was not significant.

Cyclic nucleotide data expressed as a percentage of day

14 pregnancy levels are shown in Table 4 and Figure 6. A profound increase in cyclic AMP levels was noted on day 20 of pregnancy followed by declines on both days 1 and 3 of lactation. In contrast, cyclic GMP levels were unchanged when compared with day 14 levels and were elevated 50% during the two days of lactation measured.

When the data were expressed as a ratio of cyclic AMP to cyclic GMP, there was a noted a significant (P<0=005) in crease on day 20 (71.89) when compared with day 14 of preg nancy (30.38), day 1 of lactation (33.83) and day 3 of lactation (29.87). (See Table 5 and Figures 7 and 8).

Effect of Oxytocin

No significant changes in mammary gland cyclic GMP levels were noted following injection of oxytocin into anesthetized

lactating rats (Table 6; Figure 9)= Mean values for p mol

cyclic GMP per g wet mammary tissue were 23.02, 19,14, 17,52

and 17.60 for controls and 30, 90 and 300 second post in

jection, respectively. Table 2. Mamraciry gland cyclic nucleotide content

No. ïîWT^ cAîIP . cGMP V Total cAMP Total cGMP of (g) (p mol/gn (P mol/g ) (p mol) (p mol) rats

P14 9 3.12+0. 19° 143.33+19. 14 5.36+0. 55 451.89+72.23 16.64+1. 74

P20 8 3.70+0. 34 390. 50+35.04 5.70+0. 52 1490.34+238.96 20.89+2. 41

LOI 9 5.15+0. 13 250.78+37. 13 7.97+0. 85 1275.64+189.53 41.30+4. 88 00 L03 9 4.92+0. 38 217.11+38. 01 8.07+0. 81 1069.68+224.55 w 13+3.24

^Total tissue wet weight of 3 abdominal-inguinal mammary glands.

^Picomoles/gm wet tissue.

^Mean + S.E.M. 44

400

8.0

• 300 1 1 1 Ui • 3 UJ CO 7.0 3 CO (0 {~ CO H- H ^ 2001 UJ 5 o o OL \ z 6.0 a. < z o o o CO iii ly -J _j O 100 o r z o o u 5.0 Z

20 I 3 PREGNANCY —LAC-

Figure 4. Picomoles of cyclic nucleotide per gram of wet mammary tissue Table 3. Mammary gland cyclic nucleotide^content per milligram DNA

Total CAMP Total cGMP Total DNA cAMP/DNA cGMP/DNA (p mol) (p mol) (p mol) (p mol/mg) (p mol/mg) n M i P14 451.89+72.23* 16.64+1. 74 8.47+0. 41 C 94+7.23 1.98+0. 20

P20 1490. 34+238.96 20.89+2. 41 11.13+0. 53 132, 21+19.14 1.88+0. 20

LOI 1275.64+189.53 41.30+4. «8 12.61+0. 48 100.53+13 .56 3.25+0. 33

L03 1069.68+224.55 38.18+3. 24 13.44+0. 53 81.39+17 .19 2.84+0. 21

^Mean + 46

I I 6 < z !.. o o z v a. z T/ o u z CL

i a i _i i 1 14 20 1 3 PREGNANCY — LAC

Figure 5. Picomoles (pm) of cyclic nucleotide per milligram (mg) DNA 47

Table 4. Cyclic nucleotides as a percent of pregnancy day 14 levels

Percent Percent , (pm/gm)^ (pm/mg DNA) cAMP cGMP cAMP cGMP

P14 100.00 100.00 100.00 100.00

P20 272.45 106.14 249.73 95.43

LOI 174.97 148.42 189.89 164.97

L03 151.47 150.28 153.74 144.16

^Picomoles cyclic nucleotide/g wet tissue.

^Picomoles cyclic nucleotide/mg DNA.

Table 5. Cyclic nucleotide ratios

cAMP/cGMP (mean)

P14 30.38+5.44®

P20 71.89+8.60

LOI 33.83+5.59

L04 29.87+6.20

^Mean + S.E.M. 48

PM/G WET TISSUE

250 - M PM/MG DNA

(O _j 200 UJ > UJ

E >- < o u_ o O' û. z < o

pgo LOI LÔ5 PZU Lui LUÔ

Figure 6» Percent change of cyclic nucleotides when com pared to levels on day 14 of pregnancy (P14 = 100%)

I 49

100

50 oc a. z o o r âl z 258- < o I 1

PI4 P20 Lui L05

Figure 7. Cyclic AMP;cyclic GMP ratio 50a

PM CAMP/G WET TISSUE PM CGMP/M6 WET TISSUE

P20 0.001 N.S. P20 LOI 0.025 0.025 LOI 0.025 0.05

L03 N.S. 0.005 N.S. L03 0.025 0.05 N.S.

PI4 P20 LOI PI4 P20 LOI

CAMP/C6MP RATIO

P20 0.001

LOI N.S. 0.005

LOS N.S. 1 0.005 N.S. 1

PI4 P20 LOI

PM CAMP/MG DNA PM CGMP/MG DNA

I Z I P20| 0.005 I P2o|^N.S. . 1 Lw0! I 0.005 I N.S. I LOI i 0.005 i 0.005 !

L03 N.S. 0.05 N.S. LOs! 0.01 1 0.01 N.S.

PI4 P20 LOI PI4 P20 LOI Figure 8. Significance levels (Student's t-test) between groups for cyclic nucleotide analysis 50b

Table 6. Effect of exogenous oxytocin of mammary gland cyclic GMP levels of lactating rats

Cyclic GMP (p mol/mg)a

Control 23.02 + 4.47°

30 sec° 19.14 + 3.61

90 sec 17.52 + 2,47

300 sec 17.60 + 2.72

^picomoles/mg wet tissue.

^Mean + S.E.M.

®Time elapsed after oxytocin injection

•o MEAN OXYTOCIN

SECONDS Figure 9. Effect of exogenous oxytocin of mammary gland cyclic GMP levels of lactating rats 51

DISCUSSION

Changes in rat mammary gland cyclic AMP levels during pregnancy and lactation have been reported by Sapag-Hagar and

Greenbaum (1973). Increasing levels during pregnancy, a precipitous drop around the time of parturition, and de creasing levels during lactation suggest that cyclic AMP may be important in suppressing events associated with lactogenesis and lactation.

Recent studies (Goldberg et al. 1973a,b) suggest that cyclic GMP may be important in controlling cellular events antagonistic to those mediated by cyclic AMP. This investi gation was designed to explore the relationship between these two cyclic nucleotides in the rat mammary gland around the time of parturition and lactogenesis.

The results demonstrate a distinct change in the ratio of cyclic AÎ'!? to cyclic GM? on day 20 of pregnancy which im

mediately precedes by one day the reported increase in lactose

content (Yokoyama et al. 1969) and lactose synthetase activity

(Kuhn 1968)-in rat mammary glands. It seems reasonable to think

that this shift in the cyclic nucleotide ratio might have a

relationship to the onset of lactogenesis. The changes in

cyclic AMP levels from day 14 of pregnancy to day 3 of lac

tation follow very closely the pattern reported by Sapag-Hagar

and Greenbaum (1973) although the levels reported here were 52 found to be consistently lower. Furthermore the decrease in cyclic AMP levels noted between day 20 of pregnancy and day 1 of lactation occurs over the same time period that Majumder and

Turkington (1971b) reported a drop in cyclic AMP-dependent protein kinase activity ^ vivo in mouse mammary glands.

The increase in cyclic GMP levels noted between day 20 of pregnancy and day 1 of lactation may be an antagonistic event, opposed to or contributing to the decrease in cyclic AMP levels over this same time period.

Neither prolactin (Majumder and Turkington 1971b) nor

glucocorticoids (Thompson and Lippman 1974), both of which

are necessary for lactogenesis and lactation, are known to

have any direct effect on cyclic nucleotide metabolism.

Cyclic AMP does, however, seem to be involved in the activa

tion of a protein kinase from mouse mammary gland which pre

cedes prolactin-induced phosphorylation of specific nuclear

proteins (Majumder and Turkington 1971b). Prolactin has

also been shown to induce the synthesis of both A and B

proteins of lactose synthetase. The B protein (a-lactalbumin)

remains at low levels until the onset of lactation. Possibly#

the increase in cyclic AMP and cyclic AMP-dependent protein

kinase are involved in the differentiation of mammary epi

thelial cells and the induction of a-lactalbumin directly

before the onset of milk secretion. After differentiation,

cyclic AMP and cyclic AMP-dependent protein kinase may no 53 longer be necessary for the function of the lactational cell.

(In fact, it has been shown that cyclic AMP inhibits the ac tivity of several metabolic enzymes in the rat mammary gland

(Sapag-Hagar et al. 1974), including fatty acid synthetase which has been shown to be elevated during lactation.)

The finding that cyclic AMP inhibits fatty acid synthesis is supported by Bricker and Levey (1972) who reported a sig nificant inhibition of fatty acid and cholesterol synthesis in rat liver slices by cyclic AMP (5 x 10*"^ M), although this study and the study by Sapag-Hagar et al. (1974) using 10~^M dibutyryl cyclic AMP employed the cyclic nucleotide in extreme excess of physiological concentrations. In addition, Allred and Roehrig (1973) demonstrated that cyclic AMP inhibits acetyl

CoA carboxylase activity in rat liver, the report by Carlson and Kim (1973) that partially purified acetyl CoA carboxylase can be inactivated by a phosphorylation reaction in the presence of ATP, Mg^^, and a protein fraction ("Fraction K") suggests a

possible control point for a cyclic AMP-dependent kinase. Al

though cyclic AMP was reported to have no effect on enzyme ac

tivity, a cyclic AMP-dependent protein kinase cannot be ruled

out since the protein "Fraction K" was not characterized, Acti^

vation of a protein kinase (by high cyclic AMP levels) responsi

ble for phosphorylation and inactivation of acetyl CoA carboxyl

ase would be counter to the ^ novo synthesis of milk tri- 54 glycerides during lactation.

The rapid decline in cyclic AMP levels, noted here and elsewhere (Sapag-Hagar and Greenbaum 1973), at the time of parturition and lactogenesis and the continued decline through out lactation (Sapag-Hagar and Greenbaum 1973) are in accord with a repressive role of cyclic AMP on fatty acid synthesis.

Cyclic GMP may play an important role in causing the decline in cyclic AMP levels noted from day 20 of pregnancy to day 1 of lactation, since adenylate cyclase activity has been reported to be enhanced by the presence of GTP. Theoret ically, then, an increase in guanylate cyclase activity would increase the conversion of GTP to cyclic GMP and would deplete the pool of cyclic GMP capable of interacting with the adenylate cyclase. Therefore,a decrease in GTP would also cause a decrease in the cyclic AMP output of the cell.

Ionic fluxes within the mammary epithelial cell may be partly responsible for changes in the levels of cyclic nucleotides at the onset of lactation. Ca has been shown to in hibit adenylate cyclase activity and to stimulate guanylate

cyclase. The probable presence of increased intracellular

Ca** during lactation could inhibit adenylate cyclase activity.

Prolactin has been observed to increase the transport of ions

across intestinal mucosa (Mainoya 1975) and, although the same

effect has not been investigated in mammary epithelial cells,

it seems to be a reasonable possibility. 55a

Recently, oxytocin has been demonstrated to increase cyclic GMP levels in rat uteri (Goldberg et al. 1973b). Per haps an increase in the levels of circulating oxytocin at par turition and in response to suckling during lactation ate re sponsible for the elevated levels of cyclic GMP in the mammary gland during lactation.

The preliminary test of this hypothesis reported here suggests that exogenous oxytocin is not involved in raising cyclic GMP levels within the mammary gland. However, more investigation is needed before completely ruling out the role of oxytocin. The 2,5 to 3-fold greater values for cyclic GMP assayed during this experiment suggest possible differences in technique (sodium pentobarbitol anesthesia) which I should reinvestigate before drawing conclusions.

It is hard to determine from these data if cyclic nucleo tides play any significant rcls in mammary gland cell prolifer ation since cyclic AMP levels were observed to be both in creasing and decreasing at the same time that DNA wqs in creasing, Sapag-Hagar and Greenbaum (1973) essentially re ported the same results but subsequently reported (Sapag- -3 Hagar et al« 1974) that very high concentrations (10 M) of dibutyryl cyclic AMP significantly inhibited DNA and DNA syn thesis in rat mammary expiants. The high concentration of cyclic nucleotide used in their study, makes interpretation of the role of physiological levels of cyclic AMP on the control 55b of cell proliferation difficult.

It appears from the data presented here and elsewhere

(Sapag-Hagar and Greenbaum 1973) that control of cell prolif eration in the mammary glands is not the primary function of cyclic nucleotides. Also, there is a noticeable absence of any cell proliferation during the first few days of lactation which is contrary to some reports (Greenbaum and Slater 1957;

Griffith and Turner 1961; Traurig 1967) but in agreement with

Liu and Davis (1967). This could possibly be due to strain differences in the rats used for these investigations.

Although the molecular control mechanisms of lactogenesis in the secretory cell remain obscure, the changes in cyclic nucleotide levels noted here are probably closely related to events directly preceding lactation.

The reciprocal changes in cyclic AMP and cyclic GMP re ported here for the mammary gland at the time of parturition and lactogenesis are in accord with Goldberg's "dualism theory" for the two cyclic nucleotides. Points of speculation re garding differentiation of the mammary epithelial cell into a functional lactational cell, with respect to cellular cyclic nucleotide changes, might include the following;

1. Differentiation of the epithelial cell involves the induction of proteins specific to milk such as casein and enzymes involved in lactose synthesis (galactosyl transferase and a-lactalbumin). The in duction of these proteins in mammary expants by prolactin has been reported to follow changes in the NHP fraction of nuclear proteins brought forth by 56a insulin administration and cell division and hydrocortisone-stimulated differentiation of daughter cells (Turkington et al. 1973). A continuing in crease in the amount of cyclic AMP-dependent protein kinase within the mammary tissue throughout pregnancy could possibly be due to hormonal stimulation of transcription of specific genes by prolactin during the last half of pregnancy (Majumder and Turkington 1971). Protein kinase, activated throughout preg nancy by increased levels of cyclic AMP, is probably involved in a number of tissue-specific phosphoryla tion reactions including the phosphorylation of chromatin proteins and specific cellular enzymes. The phosphorylation of chromatin protein, usually thought to be associated with depression of genes, has been shown to be involved in repression of RNA polymerase activity in certain situations (Kleinsmith 1975). Therefore, the phosphorylation of nuclear proteins by mammary gland cyclic AMP-dependent protein kinase noted by Kadohama and Turkington (1973) could possibly be involved in repressing the transcription of genes coding for lactational proteins.

High cyclic AMP levels do not appear to be compatible with the activities of several enzymes in milk synthesis. The enzymes involved in fatty acid synthesis seem to be inhibited to the greatest extent by cyclic AMP (Sapag-Hagar et al. 1974) although the supraphysiological concentrations used to demonstrate inhibition leave room for question.

Cyclic GMP and guanylate cyclase activity may be in volved in the modulation of intracellular cyclic AMP levels. These data are compatible with, although not proof of, such a role for cyclic GMP, A more complete investigation of the events occurring between day 20 of pregnancy and day 1 of lactation would allow a better interpretation of the changes in cyclic nucleo tides over this period.

Separate cyclic nucleotide-dependent protein kinases for the two cyclic nucleotides involved in the phos phorylation of separate substrates could be means of controlling differentiation especially if the specific substrates are DNA-associated proteins. 56b

5. Changes in degradation of the cyclic nucleotides by specific phosphodiesterases is a possible explanation for changing amounts of assayable cyclic AMP and cyclic GMP in the mammary glands during pregnancy and lactation. Sapag-Hagar and Greenbaum (1973) noted an increase in cyclic AMP phosphodiesterase activity throughout lactation concommitant with a decrease in cyclic AMP. On the other hand, adenylate cyclase activity paralleled the decrease in cyclic AMP during lactation. Much less is known about the activities of guanylate cyclase and cyclic GMP phosphodiesterase, but cyclic GMP levels are almost certainly dependent on the levels and rates of activity of these two enzymes within the tissue.

6. Although no clear evidence exists for direct media tion (of hormonal stimulation of the mammary gland at lactogenesis) by either cyclic AMP or cyclic GMP, there could possibly be indirect effects involving hormonal induction of specific cyclic nucleotide- dependent proteins such as the protein kinase II of Majumder and Turkington (1971a),

The role of cyclic nucleotides in controlling intracellu lar events associated with lactogenesis is unclear except for the observation that high levels of dibutyryl cyclic AMP are able to inhibit the activity of several constitutive enzymes in mammary expiants (Sapag-Hag^r et al. 1974). Even less di= rect evidence is available concerning the role of cyclic GMP in this process, however, the data, presented here suggest a possible antagonistic role for cyclic AMP and cyclic GMP in the regulation of cellular events in the rat mammary gland directly preceding lactation. 57

SUMMARY

Mammary gland levels of RNA, DNA, cyclic AMP and cyclic

GMP were assayed in rats on the 14th and 20th days of preg nancy and on the 1st and 3rd days of lactation. Mammary gland DNA levels and RNA;DNA ratios increased from day 14 of pregnancy to day 1 of lactation; no change in either parameter was noted between the 1st and 3rd day of lacta tion. Cyclic AMP levels were observed to peak on the 20th day of pregnancy and to decline from day 20 of pregnancy to day 3 of lactation. Cyclic GMP levels were low on days 14 and

20 of pregnancy and elevated on days 1 and 3 of lactation.

The cyclic AMP;cyclic GMP ratio was elevated only on day 20 of pregnancy while the ratio remained essentially unchanged on day 14 of pregnancy and days 1 and 3 of lactation. 58

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APPENDIX A: NUCLEIC ACID ANALYSIS

Removal of Lipids from Mammary Glands

1. Immediately after removal of the six abdominal-inguinal mammary glands they are immersed in 50 ml of a solution of chloroform and methanol (2:1 by volume).

2. The tissue is then minced with a scissors and kept in chloroform/methanol solution for 12 hours under constant mild agitation.

3. The solvent is poured off and fresh chloroform/methanol (50 ml) is added to the minced tissue for another 12 hour extraction, again keeping under mild agitation.

4. The chloroform/methanol is decanted off and ethyl ether (50 ml) is added to the minced tissue. Extract for 12 hours with agitation.

5. Pour off the ether and add a final 50 ml of ether and extract for another 12 hours under agitation.

6= Pour off the ether and allow to air dry over night.

7. Place the dried fat-freed tissue (DFPT) in an oven at 50 C for two days, then place in a desiccator until ready for weighing.

8. After the DPFT has been weighed, it is ground to a fine powder using a Wiley mill equipped with a number 40 sieve.

9. The powdered DFFT is placed in an oven, again at 50 C for two days to insure dryness and then placed in a desiccator until ready for weighing samples for analysis.

NOTE: Extraction times are not at all critical, change solutions in the morning and evening. Be sure DFPT powder is dry before weighing. 73

Procedure for Extracting Total Nucleic Acid

1. 25 mg samples of DFFT (dry fat-free tissue) are suspended in 4.5-5 ml of 5% trichloroacetic acid (TCA) in thick walled, 12 ml volumetric centrifuge tubes.

2. Allow the material to stand for two hours or until the tissue is saturated. Gently tap the tubes to cause the saturated DFFT to fall to the bottom of the tube.

3. Then place the tubes in a 90 C water bath for exactly 15 minutes. The tubes should be covered by glass marbles to reduce loss by evaporation.

4. After removing the tubes from the hot water bath they are immediately transferred to a cold water bath.

5. When cooled the tubes are centrifuged for 10-15 minutes at 3-4000 rpm.

6. The supernatant is decanted into another volumetric centrifuge tube.

7. The residue is resuspended with 4.5-5 ml of 5% TCA, placed in the 90 C water bath for exactly 15 minutes, and cooled as before.

8. This second digest is centrifuged, and the decant combined with the decant from the first extraction.

9. The combined extracts are then brought up to a volume of 10 ml with 5% TCA.

10, The extracts are then transferred to glass culture tubes for storage under refrigeration, until ready for analysis. 74

Preparation of Stock Hydrolyzed DNA and RNA Solutions

Stock solutions of hydrolyzed DNA and RNA are prepared containing a concentration of 1 mg/ml. From these, dilu tions can be made for determinations of standard curves.

The best procedure for preparing these is as follows:

1. Place about 80 ml of 5% TCA in a 250 ml glass beaker. Put a teflon coated magnetic stirring bar in the beaker.

2. Cover the top of the beaker with a double layer of thick aluminum foil. Poke a small hole in the center of the foil and insert a thermometer through the hole into the fluid. Place the therinometer at an angle so that it will not interfere with the stirring bar.

3. Place this apparatus on a heated magnetic stirrer and heat the TCA solution to exactly 90 C. (Make sure you can maintain this temperature for a period of 15 minutes.)

4. Pour 100 mg of DNA or RNA into the hot stirring TCA. After exactly 15 minutes from the time you put in the nucleic acid, the heaker is removed and placed in a cold water bath.

5. The solution is then put into a 100 ml volumetric flask and filled to volume vrith 5% TCA. This stock can be stored under refrigeration for up to six months.

6. To more accurately determine the concentration of these hydrolyzed standards a phosphorus content is determined as described in the Appendix, 75

Colorimetric Determination of DNA Content

Preparation of reagents

1. Diphenylamine reagent:

This reagent should be prepared on the day it is to be used. Into 100 ml of A. R. glacial acetic acid (Baker's) is dissolved 1.5 g of reagent grade dipheylamine (use only Matheson, Coleman and Bell), and 1.5 ml of concen trated H5SO4. Just before use add 0.5 ml of aqueous acetaldenyde (16 mg/ml) to the reagent.

2. Standard hydrolyzed DNA solutions:

From the prepared stock hydrolyzed DNA solution make at least five different dilutions, the largest containing no more than 500 ug of DNA.

3. IN perchloric acid:

Add 9 ml of concentrated (70%) perchloric acid to 91 ml of glass distilled water.

Method of color development

1. 1 ml aliquots of nucleic acid extracts, as well as standards, are placed in test-tubes. To each tube is added 1 ml of 1 N perchloric acid and 4 ml of di phenylamine reagent.

2. A blank is prepared by combining 1 ml of 5% TCA with 1 ml of 1 N perchloric acid and 4 ml of diphenylamine reagent.

3. Color is developed in the dark at room temperature for 16-20 hours.

4. The absorbance is measured at 600 ma. 76

Calculations

A standard curve is prepared by plotting the optical densities of the five DNA standards against their concentra tions. From this, the concentration of DNA in each of the extracts can be determined. Since standards and extracts were treated the same, no dilution corrections are necessary.

Colorimetric Determination of RNA Content

Preparation of reagents

1. Orcinol reagent:

Orcinol should be a white crystalline product. Only orcinol purchased from Calbiochem was found suitable for use without purification.

A solution of 0.5 g of FeClo per 100 ml of concentrated HCl is prepared, which can oe stored indefinitely. Immediately before use, 1 g of orcinol is added per 100 ml of solution.

2. Standard dilutions of both the hydrolyzed DNA and RNA stock solutions are required. Have five dilutions of each, the largest dilution having no more than 500 ug DNA or RNA.

Method of color development

1. 0.5 ml aliguots of nucleic acid extracts, as well as 0.5 ml aliquets of both DNA and RNA standards, are placed in test-tubes. To each test-tube is added 2 ml of 5% TCA and 2.5 ml of orcinol reagent.

2. A blank is prepared by mixing 2.5 ml of 5% TCA with 2.5 ml of orcinol reagent. 77

3. Place tubes in a hot water bath (95 C) for exactly 20 minutes. Have marbles on the tubes to prevent loss from evaporation.

4. Cool the tubes in a cold water bath.

5. The absorbance is measured at 660 mu.

Calculations

Two standard curves must be prepared; one for the DNA standards and one for the RNA standards. Orcinol reacts with

DNA as well as RNA, although to a much lesser extent. Never theless, in each sample a correction must be made for the fraction of the total optical density measured which was due to the presence of DNA in the extract. This is done as follows:

1= A standard curve is prepared plotting the optical densi ties of the DNA standards against their concentrations.

2. Having previously determined DNA by the diphenylamine method, the amount of DNA present in each of the 0.5 ml aliguots can be calculated. This means a separate calcu lation must be made for each sample.

3. From the above standard curve it can be determined how much optical density will result from the amount of DNA in the aliquot.

4. This calculated optical density due to DNA must be sub tracted from the total optical density obtained from! the spectrophotometer. The remaining optical density, then, is that due to the RNA present in the 0.5 ml aliquot. (The "corrected" optical density.)

5. A standard curve is made plotting optical density of the RNA standards against their concentration. 78

6. Using this standard curve the concentration of RNA in each sample can be determined from the "corrected" optical densities.

Procedure for the Analysis of Nucleic Acid Content by Colorimetric Determination of Phosphorous

Preparation of reagents and solutions

1. Standard phosphate:

A stock solution of monopotassium phosphate is made which contains 80 ug of phosphorus per ml. Into a liter volu metric flask dissolve 0.3509 g of monopotassium phosphate in water, add 10 ml of 10 N sulfuric acid, and dilute to the mark with distilled water. (It is suggested that glass distilled water be used throughout the procedure.) Make four dilutions from the stock; 40 ug/ml, 20 ug/ml, 10 ug/ml, 5 ug/ml.

2. Molybdate (ACID) reagent:

Dissolve 25 g of ammonium molybdate in 200 ml distilled water. Rinse into a liter volumetric flask containing 500 ml of 10 N Sulfuric acid, and dilute to mark with water. Store in a wax lined bottle under refrigeration.

3. Molybdate reagent:

Dissolve 25 g of ammonium molybdate in a liter of dis tilled water. Store in a wax lined bottle under refriger ation.

4. Amino-napthol-sulfonic acid reagent (ANS):

This reagent can be bought pre-mixed in dry form from Harleco (Hartman-Leddon Company), item number 52532. Prepare according to manufacturer's instructions.

Prepare stock solutions of 5 N and 10 N. 79

Determination of standard curve

Color development of the standard phosphate solutions is carried out in 50 ml volumetric flasks according to the following procedures;

1. Add 5 ml of standard phosphate solution.

2. Add 30 ml of distilled water.

3. Add 5 ml of molybdate (ACID) reagent.

4. Add 2 ml of ANS reagent.

5. Mix - dilute to mark with water - mix again.

6. Let stand for 5 minutes and immediately measure against a blank at 700 mu.

A blank is prepared in exactly the same way, except that the

5 ml of standard phosphate is replaced by another 5 ml of dis tilled water.

From this, a standard curve relating phosphorous con centration to absorbance can be made.

Determination of nucleic acid phosphorus content

Assuming DNA and RNA standards have been prepared by hydrolysis in 5% TCA, the procedure for determining phosphorus content is outlined as follows:

1. Into a large lipped Pyrex test-tube (200 mm x 25 mm) add 5 ïûl of the stock nucleic acid solution to 5 ml of, 5 N sulfuric acid. 80

2. Put about three glass boiling beads in the mixture and boil under a microburner. When the solution has boiled down to about a milliliter or two, a dense fume will appear and the solution turns yellow. When this occurs, turn the flame down low but do not let the boiling stop. The solution will get progressive ly darker and when there is no further blackening, add one drop of nitric acid so that it runs down the wall of the test-tube. This should eliminate most of the color. Add another drop if necessary, but do not add an excess. Continue boiling for about 30 seconds to remove all the nitric acid.

3. Remove from heat and allow it to cool down slightly; then put it under the tap to further cool it.

(Caution) This procedure should be performed under the hood. If at any time the solution stops boiling, turn up the flame slightly while agitating the solution until boiling again begins. If you do not agitate, it will superheat and spurt out.

4. Empty the contents into a 50 ml volumetric flask. Wash the test-tube three times with 10 ml of distilled water per washing. Pour the washing into the flask.

5. Add 5 ml of molybdate reagent and 2 ml of ANS reagent.

6. Mix - fill to volume - mix.

7. Let stand for 5 minutes, then immediately measure the absorbance against a blank at 700 mu.

A blank is prepared by mixing the same reagents, replacing the

5 ml of nucleic acid solution with 5 ml of 5% TCA. The boil ing procedure can be eliminated.

Comparison of the absorbance measured in the nucleic acid solution with the standard curve will give the concentration of phosphorus. Concentrations of DNA and RNA can then be estimated by assuming that phosphorus is 10% of the weight of the nucleic acid. 81

APPENDIX B: CYCLIC NUCLEOTIDE ANALYSIS

Preparation of Tissue Samples for Cyclic Nucleotide Analysis

1. Freeze tissue in liquid nitrogen immediately after recovery and store at -76 C.

2. Weigh out 5-10 mg (cAMP assay) or 100-150 mg (cGMP assay) of tissue and homogenize in 500 yl 6% tri chloroacetic acid at 4 C.

3. Centrifuge homogenates at 1500 rpm at 4 C. for 30 minutes and recover the supernatant.

4. Extract the supernatant three times with 10 volumes of ethyl ether saturated with water.

5. Concentrate aqueous solution to dryness in a stream of air. Keep in 60-70 C. water bath during concentration.

6. Dissolve the residue in 1.0 ml sodium acetate buffer, pH 6.2.

7. Take 100 yl (cAMP assay) or 300 pi (cGMP assay) of reconstituted solution for radioimmunoassay.

NOTE; Because of the extremely small amounts of cyclic GMP de tected in the mammary tissue samples, it was thought nscssssry to tsst ths 5ss«y usxng s tissue repotted to have relatively high concentrations of this cyclic nucleotide, i.e. lung. Rat lung tissue cyclic GMP was in the range of 40-50 p mol/g wet tissue which is 5-10 times the mammary tissue levels and within the reported range for rat lung (Goldberg et al. 1973a), Therefore, to obtain an accurate assay for cyclic GMP in the mammary gland, the amount of tissue assayed was increased from approximately 50 mg to approximately 150 mg. 82a

The Assay

Standard curves are prepared from stock standard solu tions provided in each kit. The range of the standard curve for the cyclic AMP is 0.025-10.0 p mol. The range for the cyclic GMP assay is 0,1-10.0 p mol. In addition, each set of standards includes; 1) a "blank" tube, which represents 125 the amount of I-cyclic nucleotide "sticking" to the side of the assay tube in the absence of antiserum, 2) a "trace" 1 nc tube which represents the amount of I-cyclic nucleotide bound to antiserum in the absence of unlabeled cyclic nucleotide, and 3) a "total count" tube containing 100 125 yl I-cyclic nucleotide.

Assay tubes are set up in triplicate according to the following procedure:

1. Pipet sodium acetate buffer into tubes.^

2. Pipet appropriate amount (100 yl for cAMP assay; 300 yl for cGMP) of sample into tubes.^

NOTE: The total volume after steps 1 and 2 should be 300 yl. The amount of sodium acetate buffer added is dictated by step 2.

^In the cyclic AMP assay. 82b

3. Add 100 lil labeled cyclic nucleotide. Mix well.

4. Add 100 yl cyclic nucleotide antiserum. Mix well.

5. Shake rack of tubes well. Cover rack of tubes with foil,

6. Refrigerate at 2-4 C. for 18 hours.

7. Transfer tubes to an ice bath.

8. Add 2.5 ml 60% saturated ammonium sulfate to each tube. Mix using a Vortex mixer.

9. Keep tubes in ice bath for 10 minutes.

10. Centrifuge tubes at 2000 rpm at 4 C. for one hour, preferably using a swinging bucket rotor.

11. Decant off and discard supernatants. Retaining the tube at a downward angle, remove additional supernatant by swabbing with a cotton tipped appli cator,

12= The tubes are then counted for 5 min, on a solid crystal scintillation counter. 83

Calculations

1. Subtract the "Blank" cpm from cpm from each of the tubes.

2. The standard curve is calculated either on the basis of per cent of total count bound (standard counts/ total counts) vs concentration^ or percent of trace binding (standard counts/trace counts) versus con centration.2

3. The amount of cyclic nucleotide in each sample tube then read directly from the standard curve.

4. The cyclic nucleotides per milligram of tissue for each sample is calculated by the following formula: cyclic nucleotide _ amount of cyclic 1.0 ml mg tissue nucleotide in tube ml of aliquot

^ mg tissue homogen

5. Total cyclic nucleotide in 3 abdominal inguinal mammary glands is calculated.

cyclic nucleotide mg wet weight of 3 abdominal- mg tissue inguinal mammary glands

^In the cyclic AMP assay.

^In the cyclic GMP assay. 84

ACKNOWLEDGMENTS

My sincere appreciation and thanks go to my major professor Dr. David R. Griffith for his encouragement and guidance these past few years. Also many thanks to my com mittee members and preceptors Dr. James R. Redmond, Dr. Allen

H. Trenkle, Dr. Jerry W. Young, and Dr. Donald C. Beitz. I would like to especially thank Dr. Trenkle for the use of his

Gamma counter, Drs. Beitz and Young for the use of their spectrophotometer and Dr. Dolphin for the use of his centri fuge.

My parents deserve more credit than I can adequately give and I thank them dearly for my education.

My deepest appreciation belongs to Dianne and Tad who made it all worth while. .