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Biomedical subjects

R Roskoski

Publications and source records attributed to R Roskoski.

At least 55 records · Page 3Linked to original sources
PubMed1983

Increased choline acetyltransferase activity in pressure-overloaded right ventricles of guinea pigs.

Choline acetyltransferase activity, a biochemical indication of parasympathetic innervation, is increased in the hypertrophied right ventricle of guinea pigs after pulmonary artery constriction (PAC). The increase appears to be dependent on the severity and the duration of hypertrophy. This change in choline acetyltransferase activity suggests compensatory changes occur in the parasympathetic innervation of PAC guinea pigs which allows the right ventricle to maintain its level of parasympathetic innervation despite marked hypertrophy. Unlike the right ventricle, the SA node of PAC guinea pigs does not have detectable changes in choline acetyltransferase activity. This model of right ventricular hypertrophy also does not have detectable changes in baroreflex control of heart rate.

Animals↗
PubMed1983

Cyclic adenosine 3':5'-monophosphate-dependent protein kinase. Comparison of type II enzymes from bovine brain, skeletal muscle, and cardiac muscle.

The physical and chemical properties of purified catalytic and regulatory subunits of type II cAMP-dependent protein kinase from bovine brain, skeletal muscle, and cardiac muscle were compared. The catalytic subunits from all three sources were identical with respect to molecular weight (Mr = 40,000), amino acid composition, and isoelectric points. Furthermore, two-dimensional maps of their tryptic peptides were identical. The type II regulatory subunits from brain and skeletal muscle exhibited identical molecular weights by sodium dodecyl sulfate-gel electrophoresis and both undergo autophosphorylation; however, they differ somewhat in amino acid composition. Furthermore, two-dimensional tryptic peptide maps showed that 40% of the peptides differ and 60% co-migrate. Autoradiography of the peptide maps showed that the main phosphorylated peptide from these two sources also differ. The bovine brain type II regulatory subunit also differed from that of bovine cardiac muscle in the same manner. The molecular weights, amino acid composition, and tryptic peptide maps of the bovine skeletal and cardiac muscle regulatory subunits, however, were experimentally identical. These results suggest that type II protein kinases from bovine brain and muscle (skeletal or cardiac) represent distinct species which differ in their regulatory subunits but share a common catalytic subunit. The tryptic peptide map of the type I regulatory subunit from bovine skeletal muscle was very different from that of the two classes of type II regulatory subunit. There were, however, four polar peptides from neural and nonneural type II subunits as well as the type I regulatory subunit which co-migrated.

Amino Acids↗
PubMed1983

Neurochemical indices of autonomic innervation of heart in different experimental models of heart failure.

Parasympathetic neural regulation of the failing heart is impaired. In order to investigate parasympathetic mechanisms in experimental heart failure, measurements were made of choline acetyltransferase (CAT) activity and [3H]-quinuclidinyl benzilate (QNB) binding in hearts of 1) hamsters with skeletal and cardiac myopathy, 2) dogs with pulmonary artery constriction and tricuspid avulsion, and 3) guinea pigs with pulmonary artery constriction. Tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) activities and norepinephrine levels served as indices of sympathetic innervation. In myopathic hearts, total CAT activity decreased (P less than 0.05) compared to age-matched controls. In canine and guinea pig right heart failure, total CAT activity was normal in contractile and specialized tissues. Alterations in [3H]-QNB binding paralleled CAT activity being decreased (P less than 0.05) only in myopathic hearts. In all three models, indices of sympathetic innervation were altered in ways qualitatively different from parasympathetic indices; TH and DBH activities were increased (P less than 0.05) in myopathic ventricles, decreased (P less than 0.05) in hypertrophied canine and guinea pig ventricles and non-hypertrophied canine ventricles, and normal in non-hypertrophied guinea pig ventricles. These results indicate that alterations in cardiac parasympathetic indices vary depending on the etiology of heart diseases and differ qualitatively from alterations in sympathetic indices. Selective determinants are necessary to explain the varied changes.

Animals↗
PubMed1983

Tyrosine hydroxylase inactivation following cAMP-dependent phosphorylation activation.

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is activated following phosphorylation by the cAMP-dependent protein kinase (largely by decreasing the Km of the enzyme for its pterin co-substrate). Following its phosphorylation activation in rat striatal homogenates, we find that tyrosine hydroxylase is inactivated by two distinct processes. Because cAMP is hydrolyzed in crude extracts by a phosphodiesterase, cAMP-dependent protein kinase activity declines following a single addition of cAMP. When tyrosine hydroxylase is activated under these transient phosphorylation conditions, inactivation is accompanied by a reversion of the activated kinetic form (low apparent Km for pterin co-substrate, less than or equal to 0.2 mM) to the kinetic form characteristic of the untreated enzyme (high apparent Km, greater than or equal to 1.0 mM). This inactivation is readily reversed by the subsequent addition of cAMP. When striatal tyrosine hydroxylase is activated under constant phosphorylation conditions (incubated with purified cAMP-dependent protein kinase catalytic subunit), however, it is also inactivated. This second inactivation process is irreversible and is characterized kinetically by a decreasing apparent Vmax with no change in the low apparent Km for pterin co-substrate (0.2 mM). The latter inactivation process is greatly attenuated by gel filtration which resolves a low-molecular-weight inactivating factor(s) from the tyrosine hydroxylase. These results are consistent with a regulatory mechanism for tyrosine hydroxylase involving two processes: in the first case, reversible phosphorylation and dephosphorylation and, in the second case, an irreversible loss of activity of the phosphorylated form of tyrosine hydroxylase.

Animals↗
PubMed1983

Methacholine-induced decrease in the cholinergic muscarinic receptor content in the perfused working rat heart.

The regulation of the muscarinic receptor by the agonist methacholine was investigated in the perfused working rat heart. After 2.5 hr of perfusion with 4 microM methacholine, the number of (-)-[3H]-quinuclidinyl benzilate binding sites was significantly reduced (10-18%) in the right and left atria and left ventricle when compared with controls (2.5 hr of perfusion in the absence of methacholine). Scatchard analysis revealed that there was no change in the apparent dissociation constant for quinuclidinyl benzilate. Furthermore, when hearts were perfused for 2.5 hr with methacholine in the presence of (-)-scopolamine, a muscarinic receptor antagonist, no significant loss of receptor was observed. After perfusing with methacholine for 2.5 hr followed by 2.5 hr in the absence of agonist, the number of quinuclidinyl benzilate binding sites returned to control levels. These results suggest that agonist-induced receptor decreases or down regulation requires receptor activation and not just simple receptor occupancy. This decrease, moreover, is reversible. The perfused working rat heart represents a physiological system in which the mechanisms of muscarinic receptor regulation can be studied.

Animals↗
PubMed1982

Changes in parasympathetic and sympathetic neurochemical indices in hearts of myopathic hamsters.

Sympathetic neurochemical indices in heart are increased in Syrian golden hamsters with skeletal and cardiac myopathy. The possibility that parasympathetic neurochemical indices might be altered was investigated in myopathic and normal hearts by measuring activity of choline acetyltransferase involved in acetylcholine synthesis. Confirming a previous report, tyrosine hydroxylase activity increased in failing myopathic hearts and norepinephrine concentration decreased. Extending previous work, tyrosine hydroxylase and dopamine-beta-hydroxylase activities in myopathic hearts demonstrated progressive, age-related increases. These changes were associated with reduced choline acetyltransferase activity in hearts of older myopathic hamsters (180 to 300-plus days). Decreases tended to be more pronounced in hamsters with cardiac hypertrophy and fluid retention (290-360 days old). Neurochemical evidence of increased sympathetic indices (dopamine-beta-hydroxylase activity) was detected at 30 days of age. Evidence of decreased parasympathetic indices (choline acetyltransferase activity) was detected at 180 days of age and persisted through terminal phases of heart failure. This study demonstrated that there are abnormalities in cardiac parasympathetic as well as cardiac sympathetic indices in myopathic hamsters.

Aging↗
PubMed1982

Selective sympathetic neural changes in hypertrophied right ventricle.

Selective pressure overload of the right ventricle in guinea pigs resulted in early and sustained reductions in tyrosine hydroxylase and dopamine-beta-hydroxylase activities in the right ventricle. No changes in tyrosine hydroxylase activity were detected in stellate ganglia sinoatrial (SA) nodal region, atrioventricular (AV) nodal region, or left ventricle. Reductions in tyrosine hydroxylase activity in stressed right ventricle were similar regardless of duration of pulmonary artery constriction, extent of hypertrophy, presence or absence of hepatic congestion, and preservation or depletion of catecholamines. The changes may represent localized loss of sympathetic nerve fibers; factors involved directly in the process of pressure-overload-induced hypertrophy may be responsible. However, sympathetic nerves remaining in hypertrophied ventricle respond normally to cold-induced sympathetic activation. The reduction in tyrosine hydroxylase activity and the maintenance of norepinephrine turnover in residual innervation to hypertrophied right ventricle support the concept that sympathetic neural regulation of hypertrophied cardiac tissue is altered but not lost.

Animals↗
PubMed1982

Tyrosine hydroxylase and choline acetyltransferase activities in ischemic canine heart.

After coronary artery occlusion, enzymes involved in the synthesis of sympathetic and parasympathetic neurotransmitters may change disparately. We investigated this in the canine heart by measuring the activity of tyrosine hydroxylase (TH) and choline acetyltransferase (CAT) in normal and ischemic tissue. Myocardial blood flow in selected regions was measured by the microsphere technique. Dogs had either ligation of the anterior descending coronary artery (LAD) or sham ligation (S). In the ischemic zone 5 h after LAD ligation, TH activity was lower than in corresponding anterior apical zones of S dogs (5.1 +/- 1.7 vs. 13.5 +/- 2.3 nmol.g-1.h-1) (P less than 0.05) with a tendency for greater decreases in endocardium than in epicardium. In contrast, there were insignificant changes in CAT activity 2.5 and 5 h after LAD ligation. Thereafter, progressive and significant (P less than 0.05) decreases occurred in CAT activity at 25 and 170 h after LAD ligation. Thus there are early heterogeneous decreases in TH activity that correlate directly with heterogeneous deficits in blood flow. Although decreases in CAT are also heterogeneous and correlate with deficits in perfusion, these changes occur later. These results indicate differences in the effects of infarction on these biochemical indices of sympathetic and parasympathetic innervation in canine heart.

Animals↗
PubMed1981

Regional distribution of tyrosine hydroxylase and dopamine beta-hydroxylase activities in guinea pig heart.

The sympathetic and parasympathetic autonomic nervous systems regulate heart rate and myocardial contractility. Using sensitive radioisotopic assays, we examined the regional distribution of tyrosine hydroxylase and dopamine beta-hydroxylase activity, which are markers for sympathetic autonomic innervation, in specialized pacemaker and conduction tissue and in contractile heart tissue. Of the 20 regions of guinea pig heart examined, we find that the highest activities occur in the sinoatrial node and the right atrial appendage. Intermediate activity occurs in the left atrium, interatrial septum and right ventricular papillary muscle. Activities in the remainder of the heart are lower and rather uniform. Comparing the enzyme markers for the parasympathetic [17] and sympathetic system, we find that these systems have different distributions. The former, for example, is closely associated with the specialized pacemaker and conduction system including the sinoatrial and atrioventricular nodes and regions rich in Purkinje fibers. The sympathetic system, on the other hand, is associated with both contractile and conducting tissue.

Adrenergic Fibers↗
PubMed1981

Tyrosine hydroxylase activation and inactivation by protein phosphorylation conditions.

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, catalyzes the conversion of tyrosine to DOPA, Cyclic AMP-dependent protein phosphorylation conditions alter tyrosine hydroxylase activity in rat striatal homogenates. In agreement with other laboratories, we find that short-term pre-incubation (3 min) of extracts under phosphorylating conditions (Mg . ATP, cAMP) increases enzyme activity two- to tenfold over control as measured during a subsequent 15-min assay. We now report that preincubation under phosphorylating conditions for longer periods (30 min) results in a loss of activity to levels equal to or below that of the control enzyme. Addition of purified bovine brain protein kinase catalytic subunit and Mg . ATP enhances activation and increases the rate of inactivation. To demonstrate that inactivation is not associated with proteolytic degradation or irreversible denaturation, the inactivated form of the enzyme can be reactivated. The protein kinase inhibitor protein decreases the activation process and prevents inactivation of the enzyme to below control values. The sedimentation coefficient is not changed by phosphorylation conditions (S = 8.8 +/- 0.1). Although the apparent Km of the enzyme for the 6-methyltetrahydropterine (6-MPH4) cofactor is reduced (0.86 mM, control; 0.32 mM, activated), it is also reduced in the inactivated form (0.38 mM). The Ki for dopamine is increased from 4.5 microM for the control to 28 microM for the activated enzyme, whereas the inactivated form of the enzyme exhibits a Ki of 10 microM. Removal of catecholamines by gel filtration fails to alter activity and the apparent cofactor Km. Moreover, both the activated and the inactivated states persist following gel filtration. It therefore appears that the activation-inactivation process is not mediated solely by the modulation of enzyme feedback inhibition or changes in the Km for 6-MPH4. We also describe a coupled decarboxylase assay in which labeled dopamine is resolved from the precursors tyrosine and DOPA by low-voltage paper electrophoresis.

Adrenal Medulla↗
PubMed1981

Hypoxia-induced changes in parasympathetic neurochemical markers in guinea pig heart.

Exposure of animals to hypoxia produces cardiovascular changes including right ventricular hypertrophy and alterations in heart rate. The activity of choline acetyltransferase, a neurochemical marker of parasympathetic innervation, and the density of muscarinic cholinergic receptors, measured by the binding of [3H]quinuclidinyl benzilate, were analyzed in the hearts of guinea pigs exposed to hypobaric hypoxia. We found a significant increase in the activity of choline acetyltransferase in the sinoatrial node in response to hypoxia after 7 or 14 days. In addition, we found significant decreases in the muscarinic receptor density in several contractile and conducting regions. The decrease in receptor density may reflect regulation by the level of occupancy; an increased occupancy may produce a decrease in muscarinic receptor number.

Acetylcholine↗