Alterations of rabbit aortic smooth muscle cell proliferation in diabetes mellitus.

OBJECTIVE: Diabetes mellitus is a known risk factor for atherosclerosis. Because initiation and/or progression of the atherosclerotic process is associated with alterations in vascular smooth muscle cell growth and differentiation, the present studies were conducted to evaluate the effect of diabetes mellitus on the proliferative behaviour of cultured aortic smooth muscle cells. METHODS: Male New Zealand White rabbits were made diabetic with a single intravenous injection of alloxan monohydrate (100 mg.kg-1) in saline. Primary cultures of smooth muscle cells were established from thoracic aortic segments of control and diabetic rabbits and used to develop multiple cell strains. The proliferative capability of secondary cultures was determined by measurements of [3H]-thymidine incorporation into DNA, cell counts, and protein content in control and diabetic cultures. The serum dependence of cellular growth was evaluated by incubation of cultured cells in growth medium supplemented with various fetal calf serum concentrations. RESULTS: Cultures of diabetic origin incorporated thymidine to a greater extent than control cultures. Although the efficiency of cell attachment was not different between control and diabetic cells, diabetic cells had a shorter population doubling time than control cells [41.08(SEM 4.15) h v 58.08(6.79) h] and achieved higher final densities than control cultures. The serum dependence of smooth muscle cell cultures for viability and growth was different between the two groups. CONCLUSIONS: These findings support the hypothesis that diabetes induces changes in vascular smooth muscle cell proliferation which may be associated with the onset or progression of the atherogenic process observed in diabetes.

Inhibition of rat vascular smooth muscle cell proliferation by taurine and taurine analogues.

The growth of rat aorta vascular smooth muscle cells (VSMCs) was measured in the presence and absence of taurine. Concentrations of taurine as low as 0.3 mM in the culture medium inhibited the proliferation of the cells, as monitored by measuring cell count, and also inhibited the rate of DNA synthesis, as examined by measuring [3H]thymidine incorporation into DNA. However, even at the highest concentration of taurine (30 mM), the doubling time of the VSMCs was only increased by 38%. Protein content of the VSMCs was decreased by 30 mM taurine. [3H]Leucine incorporation into newly synthesized protein was not affected by the highest concentration of taurine tested (30 mM), indicating that taurine did not inhibit protein synthesis but rather decreased total protein content by inhibiting cellular proliferation. The effects of other amino acids such as alanine, glycine, and serine and of various taurine analogues such as beta-alanine, guanidinoethanesulfonic acid (GES), and isethionic acid also were tested at a concentration of 20 mM for their effects on the growth of the VSMCs. Alanine, glycine, and serine had only a minimal effect or no effect on cell count, quantity of protein, and incorporation of [3H]thymidine into DNA. GES, beta-alanine, and isethionic acid had a significant effect on cell count, protein content, and incorporation of [3H]thymidine into DNA. Beta-alanine was the only analogue tested that significantly depressed [3H]leucine incorporation into newly synthesized protein. It is concluded that taurine, GES, and isethionic acid inhibited proliferation of VSMCs but did not alter normal protein synthesis or survivability of VSMCs. In contrast, other amino acids, alanine, glycine and serine, had minimal effects on VSMC proliferation and protein synthesis, whereas beta-alanine appeared to be toxic, inhibiting both VSMC synthesis and de novo protein synthesis.

Pharmacological comparison of bPTH-(1-34) and other hypotensive peptides in the dog.

The purpose of the present study was to compare the potency, effectiveness and duration of action of synthetic bPTH-(1-34) with those of other known hypotensive peptides in the anesthetized dog. Of sixteen peptides tested in the present study only 8 were demonstrated to possess hypotensive activity. While bPTh-(1-34) was one of the least potent of the hypotensive peptides, it was equal to or greater than the other peptides in terms of effectiveness and duration of action. Of all the peptides studied, substance P and eledosin were the most potent in terms of their hypotensive action. It is suggested that perhaps substance P and eledoisin might act at a different site or through different mechanisms than do vasoactive intestinal peptide (V.I.P.), corticotropin inhibiting peptide (C.I.P.), neurotensin, xenopsin, bradykinin and bPTH-(1-34).

Propranolol withdrawal supersensitivity in rat cardiovascular tissue, in vitro.

The purpose of the present study was to determine if propranolol withdrawal supersensitivity could be demonstrated under in vitro conditions. Rats pretreated for 14 days with propranolol were studied 24, 48, 72 and 96 h after propranolol withdrawal. Right atria, right ventricle strips and thoracic aortic strips were isolated for study under in vitro conditions. Dose-response curves and geometric mean ED50 values revealed the presence of supersensitivity to the chronotropic and inotropic effects of isoproterenol in vitro at 72 and 96 h after withdrawal. No change in sensitivity was found 24 or 48 h after propranolol cessation. In like manner, supersensitivity to the vasorelaxant action of isoproterenol was demonstrated in aortic strips precontracted with angiotensin II at 72 and 96 h after propranolol withdrawal. In contrast, subsensitivity to the contractile actions of methoxamine was observed 48 h after withdrawal. These data indicate that chronic propranolol pretreatment and withdrawal results in the unmasking of supersensitivity to both beta 1 and beta 2 adrenoceptor stimulation. There would also appear to be a compensatory change in alpha-adrenoceptor-induced responses in rat aorta following propranolol withdrawal.

Alterations in the myocardial beta-adrenoceptor system of streptozotocin-diabetic rats.

Previous investigations in our laboratory revealed subsensitivity of right ventricular tissue, isolated from one month STZ-diabetic rats, to the inotropic effects of isoproterenol. The present study was concerned with the characterization of this subsensitivity phenomenon. Observations of supersensitivity to methoxamine accompanied by decreased responsiveness to glucagon without a change in responsiveness to forskolin suggested a specific effect of diabetes on pathways involving receptor-mediated activation of adenylate cyclase. Radioligand binding analysis further revealed a specific decrease in the population of the high affinity state of the beta-adrenoceptor. Since the high affinity receptor state is a necessary intermediate for adenylate cyclase activation and enhanced myocardial contractility, it is proposed that the specific decrease in the high affinity population of the beta-adrenoceptor contributes to myocardial subsensitivity to isoproterenol observed in the diabetic animals. It is further proposed that the decrease in receptor population is related to increases in circulating epinephrine levels which were evident in the diabetic animals.

Reserpine-induced supersensitivity and the proliferation of cardiac beta-adrenoceptors.

Chronic reserpine pretreatment resulted in an increase in the basal developed force generated by papillary muscles isolated from the right ventricle of the rabbit. In addition, supersensitivity to the inotropic effects of isoproterenol and a concomitant increase in beta-adrenoceptor number were demonstrated. These results indicate that reserpine-induced supersensitivity is not only associated with alterations at sites beyond the level of receptor activation but also at the level of specific postjuctional membrane receptors.

The role of calcium in supersensitivity to the inotropic effects of norepinephrine.

Experiments using electrically stimulated rabbit left atria have demonstrated that supersensitivity to the inotropic effects of norepinephrine can be induced by either chronic reserpine pretreatment or hypothermia (lowering the temperature of the bathing medium). These two experimental conditions for inducing supersensitivity were not additive implying that they shared a common mechanism of action. Norepinephrine had no significant effect on the amplitude of a potentiated contraction of the rabbit atrium when the temperature was reduced from 37 to 30 degrees C or following pretreatment with reserpine (30 or 37 degrees C). Under these same conditions the ED50 of norepinephrine on the normal contraction was reduced. It is concluded that both reserpine pretreatment and hypothermia induce supersensitivity to the inotropic effects of norepinephrine by enhancing the cellular store of activator calcium while not affecting the ability of norepinephrine to release activator calcium.

Reserpine-induced supersensitivity to the inotropic and phosphorylase activating effects of glucagon in the perfused rat heart.

Glucagon (0.125--2.0 microgram) produced a dose-dependent increase in cyclic AMP, % phosphorylase a and force of contraction in the isolated perfused rat heart. Pretreatment of animals with reserpine (2.5 mg/kg, 24 h) resulted in an enhancement of the inotropic and phosphorylase activating effects of glucagon but the effect on cyclic AMP was not altered. It is suggested that reserpine-induced supersensitivity in the rat heart occurs at a point beyond the cyclic AMP step.

Effect of extracellular Na on isoproterenol-induced cAMP levels in depolarized uterus.

In the present study, the absence of a quantitative correlation between isoproterenol induced relaxation of uterine strips and tissue cAMP levels was demonstrated by using three depolarizing media: 127 mM KCl (0 NaCl), 47.5 mM KCl (NaCl), and 47.5 mM KCl (80 mM NaCl). While the degree of relaxation by isoproterenol was similar in all three media, isoproterenol (10(-4)M) increased cAMP by 100% in Na+ free depolarizing media, by 337% in Na+ containing depolarizing medium and by 600% in normal non-depolarizing medium. After pretreatment of the tissue with 10(-5) M D-600, 10(-4) M isoproterenol increased cAMP levels by 600% in all three depolarzing media. Studies using 10(-8) M isoproterenol produced qualitatively similar results. cGMP levels did not change significantly in any of the above studies. Na+ appears to be producing its effect on isoproterenol induced increase in cAMP levels indirectly by reducing the increase in intracellular Ca2+ concentration known to occur with depolarization.

Characterization of beta-adrenoceptor subtypes in rabbit mononuclear leukocytes.

Computer analysis of [125I]iodocyanopindolol competition studies using the relatively selective beta 1-adrenoceptor antagonist, ICI 89406, and the beta 2-selective antagonist, ICI 118551, on rabbit mononuclear leukocyte plasmalemmal preparations favored a two-site model indicating that both beta 1- and beta 2-adrenoceptor subtypes were present in approximately equal numbers. In contrast, similar studies performed on rabbit cardiac sarcolemma favored a one site model consistent with the presence of beta 1-adrenoceptors. Consequently, rabbit mononuclear leukocytes may provide a useful model for studying selective modulatory mechanisms of beta 1- and beta 2-adrenoceptors.

Propranolol withdrawal-induced supersensitivity to the chronotropic effects of isoproterenol in the conscious rabbit.

The present study was undertaken to determine if chronic administration and abrupt withdrawal of propranolol would induce supersensitivity to the chronotropic effects of isoproterenol in the conscious rabbit. Eight rabbits were pretreated with propranolol (40 mg/kg/day, i.p.) for one week. Dose-response curves were obtained one week prior to pretreatment and 24, 48, 72 and 168 hours after the last dose of propranolol. Isoproterenol (0.01 to 10 micrograms/kg) was injected via the marginal ear vein and heart rate was monitored continuously by recording the ECG. During the withdrawal period no significant rebound increase in basal heart rate was noted. Geometric mean ED50 values and their corresponding 95% confidence intervals were calculated and used as an index of potency. Twenty-four hours after withdrawal of propranolol pretreatment, the ED50 value for isoproterenol was significantly greater than control indicating continued surmountable beta-adrenoceptor blockade. In contrast, 72 hours after withdrawal, the geometric mean ED50 value was significantly less than control. The maximum chronotropic response to isoproterenol was not found to be different from control at any time during the withdrawal period indicating that a change in responsiveness was not induced. These data are interpreted as evidence of a true supersensitivity to the chronotropic effects of isoproterenol in the conscious rabbit following propranolol withdrawal.

Effect of insulin replacement on streptozotocin-induced effects in the rat heart.

Streptozotocin (65 mg/kg) was used to induce diabetes in male Sprague-Dawley rats. Isolated cardiac tissue exhibited a systematic depression in atrial pacemaker function and an enhancement in ventricular function accompanied by a supersensitivity to calcium relative to control animals. beta-Adrenoceptor density was found to be significantly lowered in the treated animals. However, no change in responsiveness of the tissues to isoproterenol was observed. The systematic changes in atria and ventricle were found to be completely and partially reversed respectively, by daily administration of 4-5 units of Ultralente (U-100) insulin, whereas the decrease in beta-adrenoceptor number and supersensitivity to calcium were completely reversed. These results suggest that STZ by itself might not have toxic effects in the heart and that its effects may be overcome by chronic insulin-replacement.

Functional characterization of beta-adrenoceptor subtypes in rabbit right atria.

The advent of radioligand binding studies has allowed the classification of receptor subtypes in various tissues. However, the presence of a receptor subtype in a heterogenous tissue does not insure that the receptor has a significant physiological role. beta 1- and beta 2-Adrenoceptors have been reported to coexist in the rabbit right atria. The purpose of the present investigation was to determine the physiological role of beta-adrenoceptor subtypes in catecholamine-induced chronotropic responses in the rabbit right atria through comparison of data from functional and radioligand binding studies. Rank order of potency was determined using isoproterenol, epinephrine and norepinephrine for both chronotropic and inotropic responses in the rabbit right atria and right ventricular papillary muscles, respectively. These studies indicated that the beta 1-adrenoceptor was primarily responsible for catecholamine-induced responses. Next, the beta 1-selective antagonist, atenolol, was found to inhibit the chronotropic responses of the nonselective beta-agonist, isoproterenol, and the beta 2-selective agonist, terbutaline, to the same extent. These data indicate that terbutaline produces its chronotropic effects in the rabbit right atria through stimulation of beta 1-, not beta 2-adrenoceptors. Finally, competition studies for [125I]iodocyanopindolol and the relatively selective beta 1- and beta 2-adrenoceptor antagonists (ICI 89406 and ICI 118551, respectively) indicated that the ratio of beta 1- to beta 2-adrenoceptor subtypes is 6:1. It is concluded that while both receptors may be present in the rabbit right atria, the beta 1-adrenoceptor is the predominant subtype both in density and physiological significance, while the beta 2-adrenoceptor plays little, if any role, in the chronotropic responses induced by catecholamines.

A citywide evaluation of identification of risk factors for cardiovascular disease in emergency department patients complaining of chest pain.

The local division of the American Heart Association in Lubbock, Tex, conducted a cooperative study involving all three local hospitals to assess how effectively risk factors for cardiovascular disease are identified in patients presenting in the emergency departments with a complaint of chest pain. The charts of 250 consecutive patients with chest pain were reviewed for risk factors identified by the triage nurse and by the emergency department physician or the attending physician or both. Although the rate at which risk factors were identified was good, identification was neither complete nor comprehensive. Comparison with statistics for the general population showed that some risk factors were over-reported, while others were markedly underreported. Each of the three specialties of health care professionals stressed different risk factors, and having all three involved markedly increased the level of identification. The data provide guidelines for improving risk factor identification, and the study led to the formation of strategic alliances among the different hospitals and health care professionals that should help improve secondary prevention of cardiovascular disease within the community.

Comparison of H2 receptor agonists in rabbit atria and aorta.

In an effort to compare the H2 receptors responsible for the chronotropic and vasorelaxant effects of histamine, rabbit right atria and aortae were studied using four H2 receptor agonists and the H2 receptor antagonist cimetidine. While the efficacies of the various agonists were not similar in the two tissues, the order of potency in both atria and aortae was impromidine greater than histamine greater than 4-methylhistamine greater than dimaprit. In addition, cimetidine, an H2 receptor antagonist, inhibited the chronotropic and relaxant effects of impromidine and histamine in a consistent fashion in both atria and aorta. The results obtained in the present study are consistent with the concept that the H2 receptors in rabbit atria and aortae represent one type of receptor and not two isoreceptors as is the case for beta-adrenergic receptors in these tissues.

Characterization of the inotropic response induced by stimulation of beta-adrenergic and H1 histaminergic receptors in guinea pig left atria.

The inotropic response induced by beta-adrenergic and H1 histaminergic receptor stimulation was characterized in guinea pig left atria by obtaining dose-response relationships for isoproterenol and histamine under various experimental conditions. Conditions (hypothermia, high frequencies of stimulation, and large extracellular calcium concentrations) which enhanced the ability of cardiac muscle to develop force also increased the sensitivity of the left atrium to isoproterenol while decreasing its efficacy. On the other hand, conditions which enhanced the ability of cardiac muscle to develop force depressed the efficacy of histamine to such an extent that the sensitivity to histamine was also decreased. In addition, conditions which markedly depressed the ability of cardiac muscle to develop force also decreased the efficacy and sensitivity to histamine. The data indicate that while beta-adrenoceptor stimulation results in an inotropic response under all conditions studied, stimulation of H1 histaminergic receptors results in an inotropic response only within a narrow range of experimental conditions.