Single Injection Of Isoproterenol Research Articles

What triggers eNOS translocation to the mitochondria in the heart: Adrenergic or mechanical stress?

The translocation of eNOS from the cytosol to subsarcolemmal (SSM) and intermyofibrillar (IMF) mitochondrial subfractions has been recently reported as a key trigger of exercise training-induced cardioprotection. The cardiac response to exercise is determined by both intrinsic mechanical and extrinsic adrenergic-dependent signaling pathways. Whether eNOS translocation in response to a single bout of exercise is due to mechanical stretch of the cardiac muscle or adrenergic stress remains to be studied. The aim of this work was to evaluate the impact of cardiac adrenergic or mechanical stress on the translocation of eNOS from the cytosol to the mitochondria. Mechanical stress was obtained on isolated perfused hearts by adjusting the diastolic pressure at 5 (low-stretch) or 15 (high-stretch) mmHg. The β-adrenergic stress was induced in low or high-stretch isolated hearts perfused with 10 nM Isoproterenol for 5 min. The hearts were quick-frozen. The cytosolic, SSM, and IMF + Myofilaments subfractions were obtained by differential centrifugations. The level of eNOS was analyzed by Western blot. High-stretch tended to increase the level of eNOS in both IMF and SSM subfractions without reaching statistical significance. In contrast, Isoproterenol stimulation increased markedly the level of eNOS both in SSM and IMF in low-stretch hearts, with no additional impact of high-stretch. These results suggest that β-adrenergic receptors stimulation rather than mechanical stress of the cardiac muscle can trigger the translocation of eNOS. These results obtained ex-vivo were confirmed in vivo by treating the rats with a single injection of Isoproterenol (1 mg/kg, 30 min) that increased the level of eNOS in mitochondrial subfractions. In this preliminary work, we reported that myocardial acute adrenergic stress, rather than acute stretch, is associated with eNOS translocation from cytosol to mitochondrial subfractions. The role of the eNOS-NO pathway in the normal response of the heart to adrenergic stress remain however to be studied and will be the aim of further studies.

CARDIOPROTECTIVE EFFECT OF MORIN AGAINST OXIDATIVE STRESS AND APOPTOTIC DAMAGE IN EXPERIMENTAL MODEL OF ISOPROTERENOL INDUCED MYOCARDIAL NECROSIS IN RATS

Objective: Myocardial infarction continues to be associated with high morbidity and mortality worldwide despite the availability of current therapeutic modalities. Morin, a bioflavonoid has known to possess good antioxidant, immunomodulatory and anti-apoptotic properties in previous studies. Hence, this study evaluated the cardioprotective mechanism(s) of Morin against isoproterenol induced myocardial necrosis in rats. Rationale: In this study, the ability of Morin to protect heart against isoproterenol (ISO) induced oxidative stress and myocardial infarction was evaluated. Design and method: Male albino Wistar rats were divided into five groups (8 rats in each group) i.e., I (normal), II (ISO-control), III, IV and V (morin 20, 40 and 80 mg/kg respectively). Groups III, IV and V were treated orally with daily doses of Morin accordingly for 28 days. On 26th and 27th day, a single injection of isoproterenol was injected (85 mg/kg s.c.) at 24 h interval to induce myocardial necrosis in group II, III, IV and V. On 28th day, hemodynamic parameters were evaluated, animals were euthanised and heart was excised for measurement of various parameters. Results: In ISO-control rats, there was deterioration of hemodynamic parameters, decreased anti-oxidants levels, increased cardiac injury markers and pro-inflammatory cytokines (TNF-alpha and IL-6). Also, there was increased level of Bax, Caspase-3, p-JNK, p-38 and NF-kappaB and decreased expression of Bcl-2 and p-ERK1/2 in ISO-C group. Morin dose-dependently improved hemodynamic profile, increased anti-oxidant levels, normalized myocardial architecture and reduced inflammatory markers and apoptosis. Furthermore, immunoblot analysis of MAPK pathway proteins demonstrated the mechanism responsible for anti-apoptotic and anti-inflammatory potential of morin. Conclusions: Thus, this study substantiated the beneficial effect of Morin by virtue of its modulation of MAPK pathway in myocardial injury.

Role of MAPK/NF-κB pathway in cardioprotective effect of Morin in isoproterenol induced myocardial injury in rats.

Oxidative stress plays a major role in myocardial injury. Morin, a bioflavonoid has known to possess various biological activities in previous studies. Hence, this study evaluated the cardioprotective mechanism(s) of Morin against isoproterenol induced myocardial necrosis in rats. Male albino Wistar rats were divided into five groups (n = 8) i.e., I (normal), II (ISO-control), III, IV and V (morin 20, 40 and 80mg/kg respectively). Groups III, IV and V were treated orally with daily doses of Morin accordingly for 28 days. On 26th and 27th day, a single injection of isoproterenol was injected (85mg/kg s.c.) at 24h interval to induce myocardial necrosis in group II, III, IV and V. On 28th day, hemodynamic parameters were evaluated, animals were euthanised and heart was excised for measurement of various parameters. In ISO-control rats, there was deterioration of hemodynamic parameters, decreased anti-oxidants levels, increased cardiac injury markers and pro-inflammatory cytokines (TNF-α and IL-6). Also, there was increased level of Bax, Caspase-3, p-JNK, p-38 and NF-κB and decreased expression of Bcl-2 and p-ERK1/2 in ISO-C group. Morin dose-dependently improved hemodynamic profile, increased anti-oxidant levels, normalized myocardial architecture and reduced inflammatory markers and apoptosis. Furthermore, immunoblot analysis of MAPK pathway proteins demonstrated the mechanism responsible for anti-apoptotic and anti-inflammatory potential of morin. Thus, this study substantiated the beneficial effect of Morin by virtue of its modulation of MAPK pathway in myocardial injury.

Type II iodothyronine 5'-deiodinase in mouse brown adipose tissue is stimulated by a single injection of isoproterenol in an oil/water emulsion.

A single subcutaneous morning injection of the beta-adrenergic agonist isoproterenol (ISO) (10 mg/kg body weight) in an oil/water emulsion (70/30; v/v) caused a marked increase in the activity of the enzyme type II iodothyronine 5'-deiodinase (5'-D II) in the interscapular brown adipose tissue of BDF-1 mice. After a delay of 4 hours, the 5'-D II activity began to rise in an almost linear fashion and was increased 3-fold after 8 hours, when compared to the control values. The results indicate that this method of ISO administration may be a valid tool for artificial stimulation of brown adipose tissue in animals by beta-adrenergic agonists.

Abstract 631 ADAM12 Is An Important Regulator Of Myocyte Apoptosis Induced By β-adrenergic Receptor Stimulation

ADAM (A Disintegrin And Metalloprotease)12 is a member of a family of cell surface proteins with protease and cell-binding activities. Recent work showed ADAM12 up-regulation in human heart failure. However, the activation mechanisms of ADAM12 in the heart are obscure. We hypothesized that β-adrenergic receptors (AR) stimulation regulates ADAM12 activation in neonatal rat ventricular myocytes (NRVMs) in-vitro and after injection of isoproterenol (ISO) in-vivo. Wistar rats received a single injection of ISO (5 mg/kg) and were sacrificed 6, 24 and 72 hrs later. In comparison with controls, left ventricular function was impaired in rats 24 hrs after ISO injection and started to improve at 72 hrs. The fraction of myocytes undergoing apoptosis peaked 24 hrs after ISO injection and declined thereafter. ADAM12 protein was reduced in hearts from ISO treated animals at 6 hrs, pointing to a possible increase in ADAM12 proteolytic activity. However, both ADAM12 expression and activation were significantly up-regulated at 24 and 72 hrs after ISO injection. We therefore assessed whether ADAM12 activation was involved in myocyte apoptosis secondary to excess exposure of catecholamine. Acute stimulation with ISO (10 μM, 30 min to 3 hrs) induced accumulation of ADAM12 N-terminal cleavage product in conditioned medium, demonstrating activation of the ADAM metalloprotease activity. However, chronic stimulation with ISO for 24 hrs and 48 hrs significantly increased both ADAM12 expression and secretion. This ISO-induced ADAM12 expression/activation was mediated through β 1 -AR stimulation and was dependent on intracellular calcium elevation and protein kinase C activation. Adenoviral expression of an ADAM12 protease-deficient mutant (ADAM12DeltaMP) blocks β-AR-induced myocyte apoptosis, while transduction of NRVMs with adenovirus harboring ADAM12 significantly increased myocyte apoptosis. These data suggest that ADAM12 is a regulator of myocyte apoptosis induced by β-AR in NRVMs and may play an important autocrine role in mediating the effects of β-AR on myocardial remodeling.

Alterations in myocardial ultrastructure and protein expression after a single injection of isoproterenol.

Immunochemical and electron microscopic characterization of rat myocardium was conducted 2 h and 3 weeks after a single injection of isoproterenol in rats. The relative content of several myospecific proteins (KRP--kinase-related protein, desmin), cytoskeletal proteins (tubulin, vinculin, myosin light chain kinase--MLCK) and extracellular matrix protein fibronectin was determined by immunoblotting. Two hours after injection of 50 mg/kg isoproterenol a destruction of some cardiomyocytes, contracture of myofibrils and mild edema of intercellular space was observed. The content of all the studied proteins except KRP decreased below control levels. This situation sustained 3 weeks after injection and paralleled alterations in cardiomyocyte ultrastructure. Areas of myofibrillar contracture and lysis were noted, glycogen granules were sparse; mitochondria contained arrow-like inclusions that are characteristic for calcium overload, also huge mitochondria contacting each other by specialized intermitochondrial contacts were detected. Clumps of unripe elastic fibers in enlarged intercellular space were combined with increased deposition of collagens type I and III forming areas of fibrosis. The smaller dosage of isoproterenol (10 mg/kg) rendered no significant damage in the acute postinjection period but 3 weeks later it induced the thickening of extracellular matrix around cardiac cells and the increase in KRP and tubulin content by 26 and 32%, correspondingly. MLCK levels remained depressed throughout the experiment. The rise in KRP expression was also observed after the addition of isoproterenol to cultured chicken embryo cardiomyocytes. Obtained results indicate that even a single injection of isoproterenol creates long lasting structural alterations in cardiac muscle accompanied by the increased expression of extracellular matrix proteins and several sarcoplasmic proteins apparently involved in hypertrophic response of cardiomyocytes.

Cystatin S: Molecular biology and sympathetic innervation

The ratsubmandibular glands (SMG) are innervated by both the sympathetic and the parasympathetic branches of the autonomic nervous system which, in turn, regulate the secretory functions of the glands. Parasympathetic innervation of rat SMG is present at birth, whereas sympathetic nerve fibers reach the glands by postnatal day 5. Isoproterenol (IPR), a β-adrenoceptor agonist, induces hypertrophic and hyperplastic enlargements of rat salivary glands, and induces the expression of a number of genes, including cystatin S, a member of family 2 of the cysteine proteinase inhibitor superfamily. Cystatin S gene expression is tissue- and cell type-specific, occurs transiently during development, and is not observed in adult animals. Sympathetic denervation of adult SMG, achieved by removing the superior cervical ganglion, reduces the induction of the cystatin S gene by a single injection of IPR. In addition, recent data indicate that the β-receptors present early in the developing SMG are functional and are capable of responding to IPR by increasing the expression of the cystatin S gene, even when sympathetic nerve fibers have not reached the gland. The level of IPR-induced cystatin S mRNA remains constant until day 8, at which time a dramatic induction of cystatin S mRNA is observed. This statistically significant increase in cystatin S mRNA at day 8 was diminished, but not completely suppressed, upon sympathectomy of one day old animals. These data indicate that an intact sympathetic innervation is not requisite for induction of cystatin S gene expression by IPR in developing SMG. However, sympathetic innervation is required for the full IPR response of the cystatin S gene in these glands. Collectively, these experiments suggest that factor(s) derived from the sympathetic nervous system participate in IPR-induced expression of the cystatin S gene in the rat SMG. Biomedical Reviews 1998; 9: 33-46.

Dephosphorylation of nuclear non-histone proteins in submandibular glands of rats treated with isoproterenol.

Protein phosphatase that removed [32P]phosphate from non-histone proteins, i.e., phenol-soluble acidic proteins, more rapidly and strongly than from histone proteins was present in nuclei of rat submandibular glands, but was not associated with chromatin. Cyclic AMP (10(-4)-10(-2) mM) stimulated the dephosphorylation of non-histone proteins, but not that of histone proteins. After a single injection of isoproterenol (IPR), the dephosphorylation of non-histone proteins in rat submandibular gland nuclei increased within 15 min, reached a maximum in 30 min and returned to normal control levels within 4 h. The stimulation of dephosphorylation of non-histone proteins induced by IPR was not observed after prior treatment of the animals with dichloroisoproterenol. The dephosphorylation of histone proteins was not affected by the injection of IPR. Stimulation of beta-adrenoceptors with IPR in rat submandibular glands resulted in increase in cyclic AMP and decrease in RNA synthesis in the tissues in the first few hours after the injection. This decrease in RNA synthesis was temporary and was preceded by the increase in cyclic AMP level and in the dephosphorylation of non-histone acidic proteins in the tissues. These results suggest that protein phosphatase in nuclei plays an important part in the events controlling RNA synthesis by regulating the state of phosphorylation of non-histone acidic proteins. In addition, the phosphatase may be regulated by a function of the cytoplasmic membranes.

Effects of sialagogues on ornithine decarboxylase induction and proto-oncogene expression in murine parotid gland.

The mechanism of a sialagogue-induced increase in ornithine decarboxylase (ODC) activity and the expressions of proto-oncogenes in murine parotid gland were investigated by use of isoproterenol (IPR), carbachol (CC), and methoxamine (MTX). The results were as follows: (1) The three sialagogues had similar effects on the parotid in vivo (mouse parotid after a single injection of IPR) and/or in vitro (rat parotid explants cultured on siliconized lens paper floating on 199 medium containing IPR, CC, or MTX), the order of their effectiveness being IPR > CC > MTX. (2) Northern/dot and Western blot analyses revealed that the sialagogues elevated the steady-state levels of ODC mRNA and ODC protein to maxima at two h and six h, respectively, after stimulation. The increases were roughly proportional to those in ODC activity, suggesting that sialagogue-dependent enzyme induction is regulated at the transcriptional level. (3) The mRNAs of four of nine proto-oncogenes examined showed sialagogue-dependent increases to maxima at 30 min (c-fos) or 60 min (c-jun, c-myc, and c-src) after the beginning of stimulation. These increases were all transient, with the levels returning to the control values (without sialagogue) within 60 min. (4) The IPR-dependent elevations of ODC activity and the mRNAs of ODC, c-fos, and c-jun were inhibited by monensin, but not by polymyxin B. On the other hand, the CC-dependent increases in these parameters were inhibited by polymyxin B but not by monensin.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-Adrenoceptor Control of Peroxidase Synthesis in Nasal Glands

Endogenous peroxidase activity was observed in the cisternae of the rough-surfaced endoplasmic reticulum (r-ER), including the nuclear envelope, Golgi complex, and secretory granules of the cat's nasal glands. A single injection of propranolol resulted in a decrease of peroxidase-positive secretory granules, an increase of electron-lucent granules, and no change in the level of peroxidase activity in the r-ER and Golgi complex at either 5 or 9 hours after the injection. Continuous administration of propranolol over 7 hours led to the disappearance of peroxidase activity from the r-ER and secretory granules. These data would indicate that propranolol inhibits only the synthesis of peroxidase. Twenty minutes after a single injection of isoproterenol, the rate at which the granule content containing the peroxidase reaction product was discharged into the acinar lumen increased sharply. However, 60 minutes after the injection, the secretion rate of peroxidase-positive granules decreased. The same experiment then was repeated, but this time before the isoproterenol was injected, propranolol was administered to the cats. This time no change in peroxidase activity appeared in the acinar cells. It was concluded, therefore, that the stimulation of beta-receptors enhances the synthesis of peroxidase.

Atropine treatment induced cholinergic supersensitivity at receptor and second messenger levels in the rat salivary gland

Physiological, biochemical and morphological correlates of chronic treatment of rats with the classical muscarinic antagonist atropine for 14 days (20 mg kg-1 day-1 s.c.) were studied in submandibular salivary glands. The amount of saliva collected from submandibular glands following a single injection of isoproterenol (30 mg kg-1 i.p.) was significantly larger and had higher protein concentration in rats treated with atropine than in saline-treated animals. In the glands of atropine-treated rats a conspicuous increase in the amount of rough endoplasmic reticulum (RER) along with a decrease in the mucous volume was observed in the acinus when examined by light microscopy. Several biochemical changes were observed in an enriched plasma membrane fraction from the submandibular gland of the atropine-treated rats: (1) an increase in the number of muscarinic antagonist binding sites (31 + 3.4%), (2) a decrease in the specific activity of basal adenylate cyclase, (3) a significantly lower Vmax of the adenylate cyclase in the presence of GTP (10 microM) and varying concentrations of Mg2+ (0-22.5 mM) with no apparent change in affinity of the enzyme for Mg2+ but (4) higher magnitude of stimulation in the presence of GTP (100 microM), vasoactive intestinal polypeptide (5 microM), isoproterenol (100 microM), NaF (10 microM) and forskolin (10 microM). There was however no change in the density of beta-adrenergic receptors upon atropine treatment. In tissue slices from the submandibular glands of atropine-treated rats we found lower basal cAMP levels (decrease 29 +/- 6.9%) and no significant change in the phosphatidylinositol breakdown stimulated by carbachol (10(-6) to 10(-4) M). It appears that chronic blockade of an inhibitory muscarinic input to the adenylate cyclase system is compensated by lowered adenylate cyclase activity. Phosphoinositide metabolism is not subject to the same adaptation, suggesting that cAMP may be the pivotal second messenger in the supersensitive salivary response.

Collagen turnover in isoproterenol-induced DNA synthesis and its modification by X-ray irradiation

Collagen metabolism in mouse salivary glands during the prereplicative phase of DNA synthesis induced by a single injection of isoproterenol was compared to that in liver and kidney where the cells do not respond to isoproterenol with increased DNA synthesis. Collagen prelabeled with [ 3H]proline degraded rapidly in submandibular gland following isoproterenol injection. This was consistent whether the decrease in specific activity of prelabeled collagen or in collagen content per mg DNA was measured. Collagen synthesis following degradation increased prior to onset of DNA synthesis in both parotid and submandibular glands, and later resulted in an increase in net collagen content per gland. The increase in collagen synthesis was abolished in both glands when mice had been irradiated with X-ray or injected with cycloheximide to inhibit the onset of the DNA synthesis. These changes were not detected in either liver or kidney. Thus both degradation and synthesis of collagen seemed to be correlated to the events leading to isoproterenol-stimulated DNA synthesis of epithelial cells in vivo.

Regression of the mouse parotid gland induced with isoproterenol.

ABSTRACTAcinar cells in rodent salivary glands are induced into one cycle of cell proliferation by a single injection of isoproterenol (IPR) (Baserga, 1970). Repeated injections of animals with IPR lead these cells to a few cycles of cell proliferation and then hypertrophy, when IPR stimulated proliferative response is lost and enlargement of the glands occurs. Without further IPR injections the enlarged glands regress to a normal size (Barka, 1965; van den Brenk, Sparrow & Moore, 1970). the present communication concerns the regression, or disappearance and reappearance, of the acinar cells which respond to IPR treatment.The parotid responds more readily to IPR in respect of cell proliferation and gland enlargement than the submaxillary gland (Baserga, 1970) and so was used in all studies. to produce full cell proliferation in the parotid, male SWR mice aged 3 months were given four injections at 48 hr intervals of DL‐isoproterenol‐HC1 (Sigma, St Louis, Missouri) at the dose of 7.5 mg per 30 g body weight. In the following description day zero refers to the date of fourth IPR injection. the parotid was then dissected out from animals at various days, and carefully freed of lymph nodes and fat tissues under a dissecting microscope. DNA content per gland (extracted by the method of Scott, Fraccastoro & Taft, 1956; Sasaki & Toda, 1972) increasea with the number of IPR injections, reaching a maximum after the fourth (Furuno, Iwasaki & Matsu‐daira, 1974), and then reverted to a control level in 15 days (Fig. 1). to follow the acinar cells induced into DNA synthesis, 20 hr after a second IPR treatment animals received single injections of methyl‐3H‐thymidine (The Radiochemical Centre, Arnersham, England) (3H‐TdR) by the amount of 20 μCi per 30 g body weight and then were killed at various intervals. Measurements were made on the amount of parotid DNA and its radioactivity in each animal, from which the specific activity of parotid DNA was calculated. the specific activity decreased about seven‐fold to a constant level in 15 days (Fig. 1). the DNA synthesis stimulated by a third and a fourth IPR treatment would account for the decrease in specific activity between day 3 and day 1. an autoradiographic study gave some estimate of cell turnover in the period day 1 to day 10. the IPR‐treated mice were given seventeen injections of 3H‐TdR at 12 hr intervals from 20.00 hours on day 2 to 20.00 hours on day 10 and then

Cyclic AMP Mediated Increased Amino Acid Transport: Effect of Isoproterenol Administration

A single injection of isoproterenol (IPR) stimulates cell proliferation in rodent salivary glands after a lag period of about 24 hrs. Among the many events occurring prior to stimulated DNA synthesis, there is an early increase in cAMP levels and elevated transport of amino acids into the parotid gland. Amino acid transport is also elevated in liver and pancreas, tissues not induced to proliferate by IPR. IPR-stimulated alpha-aminoisobutyric acid (AIB) transport in parotid, pancreas and liver is augmented by prior injection of theophylline and is mimicked by dibutyryl cAMP. In all three tissues, changes in cAMP levels and subsequent increases in AIB transport appear to be closely related events. Since only the parotid gland is stimulated to grow after IPR injection, amino acid transport and growth would not appear to be directly related.

Synthesis of poly(A)-associated RNA in the prereplicative phase of isoproterenol-stimulated DNA synthesis.

Rapidly labelled RNA containing poly(A) sequences has been isolated from polysomes of mouse parotid glands. Filtration on Millipore and binding to poly(u) immobilized on Sepharose-4B, together with significant RNase-resistance (11-16%) indicated that this polysomal fraction contains large tracts of polyadenylate sequences. A single injection of isoproterenol, a procedure that causes, after a lag period of 20 hrs, a marked stimulation of DNA synthesis and cell division in salivary glands of rodents, induces a biphasic increase in the incorporation of (3H)-adenosine into poly(A)-associated polysomal RNA. The incorporation of (3H)-adenosine is already increased within 1 hour after stimulation, decreases at 3 hrs and increases again at 8 hrs, parallelling previously reported changes on chromatin template activity of mouse parotids. However, the percent poly(A) content, and the size of the poly(A)-associated RNA remain constant. Actinomycin D given 30 min prior to isoproterenol, inhibits isoproterenol-Stimulated poly(A)-rich RNA synthesis. These results demonstrate that, as part of the early molecular events of its action, isoproterenol regulates transcription of adenylate-rich RNA in mouse parotid gland.

Effect of acetylsalicylic acid on development of infarct-like myocardial necroses induced in rats by a single injection of isoproterenol

The development of infarct-like foci of necrosis and the primary development of lesions of the myocardial cells through the direct action of isoproterenol and manifested during the first hour are considerably reduced in animals receiving acetylsalicylic acid. This suggests that acetylsalicylic acid not only prevents platelet aggregation but also increases the resistance of the myocardial cells to the harmful action of catecholamines.

Stimulation of acinar cell proliferation by isoproterenol in the postnatal rat submandibular gland

AbstractDuring the postnatal development of the submandibular gland, the stimulation of DNA synthesis and mitosis by a single injection of isoproterenol, measured as the percentage of labeled cells in radioautographs, was dependent on the age of the rat. The drug had no effect on the proliferative activity in two day old rats, but stimulated DNA synthesis in older (7–42 day old) animals. In general, the degree of stimulation was directly related to the proportion of acinar cells and inversely related to the proliferative activity in the gland of control rats. An analysis of various cell types revealed that the stimulation of DNA synthesis in the gland was essentially restricted to the acinar cells in rats older than 14 days of age. In seven day old rats, the acinar cells were not stimulated, but their precursors, the proacinar cells, the terminal tubule cells and the intercalated duct cells were stimulated to synthesize DNA by isoproterenol. This suggested that isoproterenol had an effect on the transformation of precursor cells to acinar cells.

Stimulation of DNA synthesis by isoproterenol in rat submandibular gland during postnatal growth.

ABSTRACTThe post‐natal growth of rat submandibular gland and the effect of isoproterenol on this process were studied. Between 2 and 42 days of age the DNA content of the gland increased linearly but the increase in RNA and protein content was more rapid after 29 days of age. The RNA: DNA and protein: DNA ratio increased linearly with age. The proliferative activity, measured by the incorporation of tritiated thymidine, was maximum in the gland of 7‐day‐old rats. It declined steadily to a low level in 42‐day‐old rats. A single injection of isoproterenol had no effect on thymidine incorporation in 2‐day‐old rats. The drug, however, stimulated DNA synthesis in older animals and the degree of stimulation was inversely correlated with the proliferative activity in control rats. Small doses of isoproterenol given to rats for 4 days between 2 and 5 days of age produced a hypertrophy of the submandibular gland. The same treatment between 7 and 10 days of age caused both hypertrophy and hyperplasia of the gland. It is concluded that both the regulation of growth and the regulation of induced cell proliferation are a function of cellular differentiation and that cell proliferation can be induced only in cells that reached a certain degree of differentiation.

Studies on phosphoproteins of submandibular gland nuclei isolated from isoproterenol-treated rats

Rat submandibular gland nuclei incubated with γ- 32P-ATP incorporated the label into histone and non-histone phosphoproteins. The latter was the predominantly radioactive fraction. After a single injection of isoproterenol (Ipr), the incorporation of 32P into non-histone phosphoproteins decreased during the first few hours, followed by an increase at 4 h which reached its peak at 24 h at a higher level compared with normal controls. The values returned to the control level at 40 h after the injection. The changes were reflected in the initial rates as well as the total level of incorporation of 32P into the phosphoproteins. Temporally, the onset of increase in the phosphorylation of non-histone phosphoproteins appeared to precede that in RNA synthesis, although peak activity of the phosphorylation coincided with the peak of RNA synthesis. The non-histone phosphoproteins which depicted maximal changes in response to Ipr were further characterized as phenol-soluble acidic phosphoproteins. The phosphorylation of histone phosphoproteins also declined after the injection of Ipr, but the recovery of the rate of phosphorylation was not observed until 16 h after the injection, reaching the control levels at 24 h. Treatment of rats with actinomycin D or cycloheximide, prior to Ipr, abolished the increase in phosphorylation of non-histone phosphoproteins observed at 24 h after Ipr. Further, the changes in the phosphorylation of nuclear phosphoproteins induced by Ipr were blocked by prior treatment of the animals with dichloroisoproterenol. The results suggest that the phosphorylation of the non-histone phosphoproteins plays an important role in the events controlling the synthesis of RNA which precedes the replication of DNA and cell. In addition, the regulation of the metabolism of nuclear phosphoproteins may be controlled through a function of the cytoplasmic membrane.

CHANGES IN CHROMATIN TEMPLATE ACTIVITY AND THEIR RELATIONSHIP TO DNA SYNTHESIS IN MOUSE PAROTID GLANDS STIMULATED BY ISOPROTERENOL

Chromatin template activity of mouse parotid glands increases after a single injection of isoproterenol (IPR), a procedure that causes, after a lag period of 20 hr, a marked stimulation of DNA synthesis and cell division in salivary glands of rodents. The increase in chromatin template activity occurs as early as 1 hr and peaks between 6 and 10 hr after IPR, paralleling previously reported changes in the incorporation of uridine-(3)H into total cellular RNA of mouse parotids. Template activity was measured in vitro in a system in which parotid gland chromatin was incubated with an exogenous RNA polymerase isolated from Escherichia coli. Similar results were obtained when template activity of parotid gland chromatin was assayed using an homologous RNA polymerase from mouse liver. Chromatin template activity in mouse parotids was also studied after the administration of drugs capable of inducing in salivary glands both DNA synthesis and secretion or secretion alone. The results indicate that the increased chromatin template activity occurring 6 hr after IPR is related to the subsequent onset of DNA synthesis. Furthermore, the increased chromatin template activity caused by IPR is inhibited by the previous administration of puromycin, an inhibitor of IPR-stimulated DNA synthesis.