Experimental cardiac hypertrophy induced by isoproterenol in the rat.

Isoproterenol (IPR) administered to rats in a dose of 5 mg/kg for seven days induces cardiomegaly. To determine the degree of myocardial enlargement, wet and dry heart weight, myocardial RNA, DNA, protein content and protein synthesis were measured. Wet and dry heart weight, and the level of cardiac RNA, DNA and protein were augmented by IPR treatment, with RNA increasing more than DNA and protein. Incorporation of 14C-leucine into the protein of the heart was enhanced by IPR. Thus the IPR-induced cardiomegaly may serve as a model for studying the development of cardiac hypertrophy under various conditions.     

Acute effects of isoproterenol on cellular autophagy. Inhibition in myocardium but stimulation in liver parenchyma

The influence of isoproterenol (IPR) on cellular autophagy was examined in left ventricular myocardium and in liver parenchyma of rats two hours after a subcutaneous injection of a low dose (3 mg/kg body weight). 4 animals were treated with IPR, 4 controls received Ringer solution. The average cytoplasmic volume fraction of the autophagic vacuoles (AV) was 1.6 X 10(-4) in the heart muscle of the controls. After treatment with IPR this value was reduced by 70% to 0.5 X 10(-4). This inhibition of cellular autophagy is interpreted as an initial anticatabolic reaction which might be responsible for the myocardial hypertrophy after chronic administration of IPR. An opposite influence of IPR was observed in the hepatocytes. The volume fraction of AV's increased twofold to 8.7 X 10(-4) after IPR, compared to 4.0 X 10(-4) in control animals. In the controls, the volume fraction of AV's in heart muscle was 57% of the value found in the liver. Comparing liver tissue after fixation by immersion and by perfusion, no statistically significant differences in the volume fractions and in the numerical densities of AV's were observed.     

Enhanced coupling of adenylate cyclase to lipolysis in permeabilized adipocytes from trained rats.

Digitonin-permeabilized adipocytes were used to study the coupling of adenylate cyclase (AC) to lipolysis in exercise-trained rats. Isoproterenol-(IPR) stimulated lipolysis in permeabilized cells was significantly greater in trained than in control rats. Under essentially identical conditions, the dose-response curve for IPR stimulation of AC activity in the absence of 3-isobutyl-1-methylxanthine was similar in trained and control rats. However, the potency of stimulation by IPR as a percentage of the basal level was greater in trained rats. AC activity and lipolysis in the presence of 3-isobutyl-1-methylxanthine were also significantly greater in trained than in control rats. Least-squares analysis by plotting the log AC vs. lipolysis values showed that the regression coefficient was about three-fold greater in trained than in control rats. The concentration of endogenous adenosine 3',5'-cyclic monophosphate (cAMP) needed to produce a half-maximal lipolytic response was 18.58 and 10.81 pmol.min-1.10(6) cells-1 in control and trained rats, respectively. Thus a positive relationship existed between lipolysis and AC activity, with a tighter coupling in trained rats. Lipolysis in response to exogenous cAMP tended to be greater in trained than in control rats, and the difference was statistically significant for 50 microM and 10 mM cAMP. Our finding support the concept that the major mechanism of enhanced lipolysis in trained rats was an increase in the activity of enzymatic step(s) distal to cAMP.     

Prevention of isoproterenol-induced cardiac hypertrophy by beta-blocking agents in the rat.

The effect of the beta-blocking agents propranolol and oxprenolol on isoproterenol-induced cardiac hypertrophy has been investigated in the rat. To evaluate the degree of inhibition biochemical parameters related to cardiomegaly were measured. When given alone in the dose range used for protection, the beta-blocking agents did not cause any significant change in total myocardial RNA, DNA and protein. Depending on the dose applied, propranolol and oxprenolol prevented partially or totally the cardiomegaly induced by isoproterenol.     

Regulation of phosphofructokinase-1 on submandibular salivary glands of rats after isoproterenol administration

The purpose of this investigation was to study the effect of isoproterenol (IPR) treatment on the regulation of phosphofructokinase-1 of submandibular salivary glands of rats. The animals were divided into control and experimental groups. In the first set of experiments, the rats received 5 mg of IPR/kg b.w. and were sacrificed at 24 hours after 1, 2, 3 and 4 doses. The content of fructose-2,6-bisphosphate (Fru-2,6-P(2)) and the activity of 6-phosphofructo-2-kinase (PFK-2) (active and total) were determined. The Fru-2,6-P(2) content was found to be reduced and the activity of PFK-2 (active and total) showed differences from the control. The active/total ratio, was higher for the group of one dose sacrificed 12 hours after the agonist injection as compared to the control. In the other groups, there were reductions which varied from 25 to 33%. In the second set of the experiment, the animals were injected with 23.0 mg of IPR/kg b.w. and were sacrificed from 5 up to 720 minutes after the administration of the agonist. After the sacrifice, salivary gland samples were analyzed for Fru-2,6-P(2). Again, a reduction in the metabolite content was observed. Using beta and alpha receptor blockers, it was found that both inhibited only partially the effect of IPR. The purification of PFK-1 up to homogeneity, from submandibular glands of rats which received 5 mg of IPR/mg b.w. as well as from the control, was performed and the Km and state of phosphorylation were determined. Rats from the group sacrificed 12 hours after the injection of the agonist showed the lowest Km for Fru-6-P. Animals which received 3 doses of IPR showed the highest phosphate content/mol of enzyme. Experiments of dephosphorylation of the purified PFK-1 from this latter group revealed that the presence of the phosphate groups influence the kinetic properties of the enzyme.     

Effect of postnatal anoxia on seizure susceptibility in rats: bicuculline-induced seizures and pretreatment with iproniazid

The effects of anoxia at 10 days of age on seizures induced by bicuculline (BIC), a specific antagonist of gamma-aminobutyric acid (GABA), and BIC-induced seizures after pretreatment with iproniazid (IPR), a monoamine oxidase inhibitor, were investigated in male adult rats. The rats with postnatal anoxia (Group A) were significantly more susceptible to BIC than the control rats (Group C). On the other hand, BIC-induced seizures in both groups were suppressed by IPR pretreatment. Especially, the suppression of BIC-induced seizures which was made by pretreatment with IPR at small doses was more apparent in Group A as compared with Group C. The present study suggests that the enhanced seizure susceptibility of adult rats with postnatal anoxia involves brain monoaminergic system.     

Expression and induction by beta-adrenergic agonists of the cystatin S gene in submandibular glands of developing rats.

Repeated administration of the beta-adrenergic agonist isoprenaline (isoproterenol, IPR), which produces hypertrophic/hyperplastic enlargements of rat submandibular and parotid glands, induces synthesis of a secretory protein shown to be a cysteine proteinase inhibitor, rat cystatin S. In the current study, Northern blot and hybridizations in situ were carried out to establish the developmental and beta-adrenergic regulation of the expression of the cystatin S gene. Cystatin S mRNA was not detected in submandibular glands of 20-day-old fetuses, nor in the glands of newborn or 10-day-old rats. However, steady-state levels of cystatin S mRNA increased between 21 and 28 days, reaching a conspicuously high concentration at 28 days; cystatin S mRNA then declined rapidly to a barely detectable level in glands of 32-day-old rats. IPR administration for 4 days induced high levels of cystatin S mRNA in submandibular glands of developing and adult rats. In both prepubertal and mature animals, induction of cystatin S mRNA in submandibular glands was more pronounced in female than in male animals. Hybridizations in situ revealed cystatin S mRNA only in acinar but not in duct cells of the submandibular gland. Developmentally, expression of the cystatin S gene coincided with acinar cell differentiation. These data suggest a complex neural, hormonal and developmental regulation of salivary cystatin genes.     

Effect of an exercise regimen on development of hypertension in rats

The possibility that a forced exercise regimen might prevent the development of hypertension induced in rats both by renal encapsulation and chronic administration of deoxycorticosterone acetate (DOCA) and NaCl has been studied. In renal hypertensive rats, forced exercise at 0.4 to 1.25 miles/day, 7 days/wk for 16-22 wk failed to prevent the development of hypertension and cardiomegaly and reduced renal concentrating ability accompanying the hypertension. In DOCA-treated rats (10 mg/wk), forced exercise at 0.4 and 0.8 mile/day, 7 days/wk for 16 wk also failed to prevent both the development of hypertension and cardiomegaly. A review of data of others reveals that exercise may delay the development of hypertension in both Dahl salt-sensitive and spontaneously hypertensive (SHR) rats and may modestly reduce the maximal level of pressure attained. Of the four models of hypertension studied to date in rats, the Dahl salt-sensitive strain appears to be the one that responded best to exercise, although blood pressure eventually reached that of sedentary controls.     

Metyrapone block of thyroid-induced cardiomegaly

Cardiac wet and dry weight, adrenal weights, blood pressure and food consumption were measured in control male rats, rats fed 0.2% dried thyroid powder in the food, rats fed metyrapone (an inhibitor of adrenocortical 11-hydroxylase, Metopirone CIBA, 50 mg/rat per day) and both metyparone + dried thyroid. Metyparone markedly inhibited thyroid-induced cardiomegaly as well as the increase in adrenal weight. Myocardial dry weight and blood pressure differences between the various groups were not significant. The results supply further evidence of the existence of a relationship between the adrenal cortex and thyroid-induced cardiomegaly. Metyrapone also completely inhibited the post-thyroid drop in body weight.     

Immunocytochemical localization of a developmentally regulated, isoproterenol-inducible protein (LM protein) in rat submandibular gland

Administration of the beta-adrenergic drug isoproterenol (IPR) produces hyperplastic and hypertrophic enlargements of the submandibular gland of the rat and induces the synthesis of specific proteins in this organ. One of these proteins, the LM (large mobile) protein, was demonstrated immunocytochemically in the submandibular glands of developing untreated and IPR-treated rats. Immunoreactive LM protein was absent in the glands of 20-day-old fetuses and 1- and 2-day-old rats. It was localized in the proacinar and immature acinar cells in the glands of 6- to 21-day-old animals, but it was undetectable at 28 days of age. In the glands of adult rats, secretory granules of the granular convoluted tubule cells showed immunostaining for the LM protein which was also present in trace amounts in the acinar cells. Daily administration of IPR for 5 days to newborn or 8- or 15-day-old rats caused an apparent acceleration of proacinar/acinar cell differentiation, and consequently it increased the frequency of cells immunostained for the LM protein as well as the amount of immunoreactive material in these cells. Thus, the expression of LM protein in the submandibular gland is developmentally regulated, and it is restricted to the stage of differentiation of proacinar cells from terminal tubule cells. IPR is capable of inducing this protein in fully differentiated acinar cells in 3-week-old or older animals.     

Changes in liver cells ploidy of young rats following isoprenaline treatment.

Cell proliferation induced by isoprenaline (IPR) stimulation in very high doses was assayed in the liver of young rats, and the formation of polyploid cells was studied form the 15th to the 70th day of life. A general stimulatory effect on a complex process of cellular multiplication, leading to a population of tetraploid cells, was found to be accelerated; the earlier appearance of binucleate cells and the subsequent significant variations in their incidence confirmed the role of this cell type as an intermediate step in the process of polyploidization. Evidence was found of concomitant size changes of the hepatocytes, which might be partially independent of the effect of DNA content. The stimulation was no longer evident 20--30 days from the end of treatment, by when the cells which had come into contact with IPR should have completed the whole sequence of events leading to the formation of tetraploid mononucleate cells.     

The effects of desipramine and iprindole on levels of enantiomers of fluoxetine in rat brain and urine

The antidepressant fluoxetine (FLU) and its N-demethylated metabolite, norfluoxetine (NFLU), each contains a chiral center. The combination of FLU and desipramine (DMI), another antidepressant, has been reported to be useful in treatment of depression, to dramatically increase plasma levels of DMI and also to produce more rapid β-adrenergic receptor down-regulation in brain than caused by DMI alone. We have now begun studies on the effects of this drug combination on the levels of FLU and NFLU enantiomers in the rat. In addition, the combination of FLU and iprindole (IPR) was also investigated. Male Sprague–Dawley rats were treated intraperitoneally with either normal saline vehicle, DMI (5 mg/kg/day), (R,S)-FLU (10 mg/kg/day) or DMI (5 mg/kg/day) + (R,S)-FLU (10 mg/kg/day) for 4 days. Following the last treatment, 24 h urine samples were collected. Rats were sacrificed and brains were removed. For the IPR study, rats were pretreated with either saline or IPR-HCl (11.2 mg/kg) and then treated 1 h later with (R,S)-FLU. After 5 h, the rats were sacrificed and brains were removed. Brain and urine samples were analyzed by gas chromatography with electron-capture detection for free (R)- and (S)-FLU and (R)- and (S)-NFLU after extraction and reaction with (?)-(S)-N-(trifluoroacetyl)prolyl chloride. The results from the brains of the rats treated with DMI/FLU indicate that levels of the enantiomers of both FLU and NFLU were significantly increased over those seen in the animals receiving (R,S)-FLU alone. In the IPR/FLU treated rats, an increase in the brain levels of both (R)- and (S)-FLU was noted when compared with rats receiving (R,S)-FLU alone; however, there appeared to be no increase in the brain levels of NFLU enantiomers. © 1994 Wiley-Liss, Inc.     

Effects of beta-adrenergic stimulation on bone-marrow function in normal and sublethally irradiated mice. I. The effect of isoproterenol on cAMP content in bone-marrow cells in vivo and in vitro.

The effect of isoproterenol (IPR) on bone-marrow cAMP content was investigated in vivo and in vitro. In unirradiated CFW mice, the bone-marrow cAMP content was found to be elevated by the administration of noradrenaline, adrenaline and isoproterenol. After IPR administration, the increase in cAMP was biphasic with maxima at 1 and 15 min. An increase in cAMP content was also noted in bone-marrow of sublethally-irradiated mice, but no further increase was observed 15 min after the administration of IPR. Elevation of cAMP by either IPR or radiation was prevented by pretreatment with the beta-adrenergic blocking agent--propranolol. IPR was also effective in increasing the cAMP content when added to suspension of bone-marrow cells. This effect was abolished by propranolol. IPR did not increase cAMP levels in bone-marrow cells isolated from irradiated animals. The results suggest that the differentiated bone-marrow cells have beta-adrenergic receptors.     

Simultaneous determination of ipratropium and salbutamol in rat plasma by LC-MS/MS and its application to a pharmacokinetic study

A novel, sensitive and specific LC-MS/MS method with silica-based solid-phase extraction was developed for simultaneous determination of ipratropium (IPR) and salbutamol (SAL) in rat plasma. Chromatographic separation was achieved on a Shiseido Capcell Pak CR column (SCX:C(18)=1:4, 150 mm × 2.0 mm, 5 μm) with a mobile phase consisting of methanol/water (85:15, v/v) containing 20 mmol/L ammonium formate and 0.1% formic acid at a flow rate of 0.3 mL/min. A tandem mass spectrometric detection with an electrospray ionization (ESI) interface was conducted via multiple reaction monitoring (MRM) under positive ionization mode. This method was validated in terms of specificity, linearity, accuracy (within ±115.4%), intra- and inter-day precision (<11.4%) over the concentration range of 8-1612 pg/mL for IPR and 50-10,000 pg/mL for SAL. In addition, stability and matrix effects of IPR and SAL in plasma were evaluated. This method has been successfully applied to the pharmacokinetic study of compound ipratropium bromide aerosol mainly containing ipratropium bromide (IB) and salbutamol sulphate (SS) after inhalation in rats.     

Effect of acute administration of isoproterenol on submandibular salivary gland of female rat

The functions of salivary glands are under the regulation of both sympathetic as well as parasympathetic nerve fibers. Further, it has also been demonstrated that chronic administration of a beta-adrenergic agonist isoproterenol (IPR) results in hypertrophy and hyperplasia of submandibular gland [Schneyer C A, Am J Physiol, 203 (1962) 232]. Specific purpose of the present attempt was to look for metabolic responses of submandibular gland of oestrous female rats at very short intervals after 10 min of administration of 5, 10 and 15 micrograms of IPR to females in oestrous condition; pharmacological action and clearance time being only 8 min. The results indicated significant reduction in case of enzymic activities of phosphorylase, total ATPase and Na(+)-K+ ATPase. Cyclic AMP-specific phosphodiesterase and succinate dehydrogenase activities were suppressed only with 5 micrograms dose, but with rising dose levels the effect was not so apparent. Protein content of the gland was reduced slightly by administration of IPR. Hence, it became clear that submandibular gland responds rapidly to IPR administration. Implications of these observations are discussed.     

Effects of aging and AT-1 receptor blockade on NO synthase expression and renal function in SHR

In an earlier study, we found increased NO production and NO synthase (NOS) expression in renal and vascular tissues of prehypertensive and adult spontaneously hypertensive rats (SHR). This study was designed to determine the effects of aging and AT-1 receptor blockade (losartan 30 mg/kg/day beginning at 8 weeks of age) on NO system in this model. Compared to the Wistar Kyoto (WKY) control rats, untreated SHR showed severe hypertension, elevated urinary NO metabolite (NO(chi)) excretion, marked upregulations of renal and vascular eNOS and iNOS proteins, normal renal function and heart weight at 9 weeks of age. Hypertension control with either AT-1 receptor or calcium channel blockade (felodipine 5 mg/kg/day) mitigated upregulation of NOS isoforms in the young SHR. With advanced age (63 weeks), the untreated SHR showed increased proteinuria, renal insufficiency, cardiomegaly, reduced urinary NO(chi) excretion and depressed renal and vascular NOS protein expressions as compared to the corresponding WKY group. AT-1 receptor blockade prevented proteinuria, renal insufficiency, cardiomegaly, and renal and vascular NOS deficiency. Thus, in young SHR, hypertension results in compensatory upregulation of renal and vascular NOS, which can be attenuated by vigorous antihypertensive therapy. With advanced age, untreated SHR exhibit cardiomegaly, renal dysfunction and marked reductions of eNOS and iNOS compared with the aged WKY rats. Hypertension control with AT-1 receptor blockade initiated early in the course of the disease prevents target organ damage and preserves renal and vascular NOS.     

ALTERATION OF CELLULAR PROLIFERATION IN THE ILEAL EPITHELIUM OF SUCKLING AND WEANED RATS: THE EFFECTS OF ISOPROTERENOL

The purpose of this study was to analyse cell proliferation in the ileum before and after neonatal closure to macromolecules and to determine the effects of the sympathomimetic amine, isoproterenol (IPR), on cell cycle parameters. 8- and 28-day-old rats were employed in this study representing the neonatal periods of pre- and post-closure ileum respectively. The duration of the cell cycle phases was determined by the percentage of labeled metaphases technique (PLM) with computerized analysis of the curves. The generation cycle time was longer in 8-day-old suckling rats (18.63 hr) as compared to the older weaned rats (11.85 hr), and most of this 6.78 hr difference was in the G₁-period (4.5 hr). Other proliferative indices were also lower in the suckling rats—mitotic index (1.9 ± 0.4% as compared to 6.7 ± 0.9%), labelling index (26.8 ± 2.5% versus 44.2 ± 2.6%) and migration rate measured as per cent labeled villus cells (8.9 ± 3.1% versus 45.2 ± 3.4%). IPR was found to inhibit cellular proliferation in the ileal epithelium of both age groups. The cell cycle of the ileal epithelium of 8-day-old rats was lengthened from 18.63 to 21.07 hr and 28-day-old rats from 11.85 to 13.98 hr. IPR produced a decrease in mitotic index from 1.9 ± 0.4% to 1.6 ± 0.4% in pre-closure ileum and from 6.7 ± 0.9% to 5.1 ± 0.6% in post-closure ileum. Labeling index decreased from 26.8 ± 2.5% to 20.0 ± 2.0% in 8-day-old rats and from 44.2 ± 2.6% to 31.1 ± 3.0% in 28-day-old rats after IPR administration. There were also significant differences in growth fraction between age groups and a significant decrease in growth fraction after IPR-treatment. From the results of this study it appears that β-adrenergic stimulation has an inhibitory effect on neonatal ileal epithelium.     

Thyroxine cardiomegaly in rats is not hypertension-bound

For 13-27 days, male rats were given dried thyroid (Thyreoidin SPOFA) in their food, 0.9% NaCl to drink instead of water, or both. In all the experiments, Thyreoidin induced pronounced cardiomegaly. The blood pressure rose only after 0.9% NaCl combined with Thyreoidin and only in experiments lasting 13 or 16 days (not 25 and 27 days). After three weeks of the experiment, therefore, hypertension disappeared, but cardiomegaly persisted. In all the experimental groups, including the controls, a significant positive correlation was found between heart weight and adrenal weight, but not between heart weight and blood pressure or between adrenal weight and blood pressure. The adrenals may thus participate in some way in the development of thyroxine cardiomegaly.     

Isoproterenol fails to produce myocardial necrosis in streptozotocin-induced diabetic rats.

Biochemical alterations in the hearts of non-diabetic and 5 weeks of streptozotocin-induced diabetic rats following isoproterenol (ISO) administration were compared. Serum lactate dehydrogenase (LDH) and myocardial adenosine triphosphate (ATP), creatine phosphate (CP), lactate and glycogen were used as indices of myocardial injury. Hearts from diabetic rats (blood glucose greater than 350 mg/dl), before ISO administration, had normal lactate levels but significantly low high-energy phosphate (HEP) levels and high glycogen levels in comparison to non-diabetic rats. No difference was observed in serum LDH levels between these two groups. ISO administration to non-diabetic rats caused myocardial necrosis as evidenced by a significant depletion of myocardial glycogen and HEPs along with significant myocardial lactate accumulation and an increase in serum LDH. However, the hearts from diabetic rats failed to show any significant HEP depletion, accumulation of lactate and leakage of LDH into serum following ISO-administration, though myocardial glycogen level was significantly lowered. These observations, in conjunction with earlier reports, point to the hypothesis that, in diabetes, there are certain alterations in the sarcolemma which hamper the process by which ISO causes myocardial necrosis.