Jerry G. Webb

Stereoselective delivery and actions of beta receptor antagonists

These studies have revealed that the delivery and actions of beta receptor antagonist drugs are controlled by a cascade of stereoselective processes involving multiple enzymes, transport proteins and receptors. In essence, the free concentration of the pharmacologically active (-)-enantiomer species of these drugs presented to cell surface beta receptors appears to be a function of the stereoselective clearance by hepatic cytochrome P-450 isoenzymes, enantiomer selective binding to alpha 1-acid glycoprotein and albumin and perhaps predominantly by stereoselective sequestration (and release) by the vesicular amine transport protein within adrenergic neurons. Stereoselectivity in the clearance of beta blocking drugs, which can favor either the (+)- or (-)-enantiomer, only appears to be important for the lipophilic drugs which are cleared by hepatic metabolism. Such stereoselectivity is due to differential stereochemical substrate requirements of individual hepatic cytochrome P-450 isoenzymes. Interindividual variations in the stereoselectivity can occur as a result of differences in the amount and expression of cytochrome P-450 isoenzymes due to genetic predisposition or other factors. In the same context, we have observed a significant correlation between the extent and stereoselectivity of binding of beta blocking drugs to plasma proteins. This is another finding which suggests that variability in the expression of individual proteins involved in the beta blocking drug-protein cascade determines the free concentration of the pharmacologically active enantiomer. However, since most observations have been made in young normal subjects, the extent of stereoselectivity in metabolism, binding and other processes is unknown in the general population where steady-state plasma concentrations can vary widely due to multiple biological factors. The observations from neural studies support the concept that adrenergic nerve endings provide a depot for the stereoselective storage and release of the active enantiomer of beta receptor antagonists. The mechanism of this release appears to involve exocytotic secretion of drug that has been stereoselectively accumulated by the neurotransmitter storage vesicles. In terms of this idea, beta receptor antagonists released during nerve stimulation may achieve concentrations of the (-)-enantiomer within the adrenergic synapse greatly in excess of those found in plasma. Such a mechanism could significantly influence both the intensity and duration of beta receptor blockade in the heart, blood vessels, brain and other target tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation of sealed vesicles highly enriched with sarcolemma markers from canine ventricle

Abstract A highly enriched sarcolemma preparation was isolated by a combination of homogenizations and differential centrifugations of a homogenate of canine ventricular tissue followed by centrifugation of a membrane fraction layered over 24% (w/v) sucrose. The membrane fragments in the preparation were found to reside in a vesicular configuration by electron microscopy. Adenylate cyclase activity, specific ouabain binding sites, ouabain-sensitive potassium phosphatase activity and high-affinity (−)dihydroalprenolol binding sites were enriched from 27- to >40- fold compared to the homogenate. 5′-Nucleotidase and antimycin A-insensitive NADH-cytochrome c reductase activities were enriched 10.1- and 3.0–3.9-fold, respectively, compared to the homogenate. Conversely, Ca2+-ATPase and succinic dehydrogenase activities were somewhat less than those expressed by the homogenate. The vesicles, or at least a fraction thereof, in the preparation were osmotically active as monitored by changes in 90° light scatter upon increases in the osmolarity of the extravesicular medium. (Na + + K + )-ATPase activity in the preparation was 23.6 and 99.9 μmol/mg per h before and after 15 consecutive freeze-thaw cycles, respectively. This suggests that the preparation contains a large fraction of intact right-side-out vesicles but that about 24% of the vesicles in the preparation may be freely permeable. Conversely, the number of specific ouabain binding sites was increased about 1.4-fold by the freeze-thaw cycles which is consistent with the presence of

Increased myocardial adenosine release in heart failure

Volume overload congestive heart failure in dogs is associated with a reduced myocardial inotropic responsiveness to the exogenous administration of beta-adrenergic agonists [10, 11]. This same blunted inotropic responsiveness to beta-agonists has now been identified in the failing human myocardium [2]. Volume overload congestive heart failure in dogs is also associated with a reduced resting coronary vascular resistance [7, 12] suggesting the possibility of increased myocardial production of a metabolic vasodilator in the failing heart. Adenosine is a metabolic coronary vasodilator [1] and also has recently been shown to antagonize the inotropic action of beta-adrenergic agonists through a mechanism involving action on the sarcolemmal adenylate cyclase system [4, 13]. Given the findings of blunted inotropic responsiveness of the failing myocardium to beta-adrenergic agonists and reduced coronary vascular resistance in heart failure, we hypothesized that heart failure was associated with elevated myocardial production of adenosine. Accordingly we measured myocardial adenosine release in normal dogs and dogs with volume overload heart failure. Basal levels of myocardial adenosine release were found to be elevated three-fold above normal in dogs with heart failure. It is possible that elevated adenosine release in the failing myocardium contributes both to abnormalities of coronary blood flow and to the blunted inotropic responsiveness of the failing heart to catecholamines.

Orthostatic hypotension with brainstem tumors

Three patients with brainstem tumors had orthostatic hypotension as the major presenting manifestation. Two patients had primary tumors that involved the dorsal medulla, pons, and rostra1 spinal cord; one was a malignant astrocytoma and the other a hemangioblastoma. The third patient had an oat cell carcinoma of the lung with subependymal spread to the medulla, pons, hypothalamus, and thalamus. Evaluation of baroreceptor function in the patient with the malignant astrocytoma showed a defect in the efferent sympathetic limb of the baroreceptor reflex arc.

Depolarization‐induced release of propranolol and atenolol from rat cortical synaptosomes

1 The accumulation and release of [3H]‐propranolol and [3H]‐atenolol were examined in synaptosomes from rat cerebral cortex. 2 Synaptosomes accumulated 20 pmol propranolol and 0.6 pmol atenolol mg−1 protein when incubated at 30°C with radiolabeled drugs (0.1 μm). 3 Exposure of propranolol‐loaded synaptosomes to elevated K+, Rb+ or Cs+ evoked a concentration‐dependent increase in propranolol efflux. The action of these ions in releasing propranolol was highly correlated with their ability to produce synaptosomal membrane depolarization, as estimated with the voltage‐sensitive dye diS‐C3‐(5). 4 Elevated K+ also promoted atenolol release from synaptosomes in a concentration‐dependent manner. 5 Veratridine (10 μm) released propranolol and atenolol from synaptosomes and these effects were antagonized by tetrodotoxin (1 μm). 6 Under Ca2+‐free conditions, K+‐induced release of propranolol was reduced by 37% and atenolol release was diminished by 68%. 7 The results support the concept that both polar and non‐polar β‐adrenoceptor blocking drugs may be accumulated by nerve endings for release by membrane depolarization and suggest that neural storage and release of these molecules may influence their concentrations at localized sites of action.

Effects of centrally administered propranlol on plasma renin activity, plasma norepinephrine and arterial pressure

Abstract Single doses of d,l-propranolol (0.1–0.5 mg/kg) administered intarcisternally (IC) to anesthetized dogs produced dose-dependent decreases in plasma renin activity and mean arterial pressure whereas identical doses given intravenously had no significant effect on these parameters. Intracisternal d,l-propranolol also significantly reduced circulating norepinephrine levels concomitant with the renin suppressing and hypotensive effects. Acute renal denervation abolished the renin suppressing, but not the hypotensive effect of intracisternal d,l-propranolol. Both d- and l-propranolol administered intracisternally significantly lowered plasma renin activity and arterial pressure to a similar degree, but intracisternal lidocaine had no effect on these parameters. These results indicate that propranolol can suppress plasma renin activity through an action within the central nervous system and suggest that both the renin suppression and hypotension produced by intracisternal propranolol resulted from a centrally mediated reduction in peripheral sympathetic activity. The equal potency of the stereoisomers of propranolol and the observed differences between the effect of propranolol and lidocaine suggest that these central actions involve a mechanism that may be independent of beta receptor blockade or the local anesthetic properties of drug.

DIFFERENTIAL EFFECT OF PERTUSSIS TOXIN ON ADENOSINE AND MUSCARINIC INHIBITION OF CYCLIC AMP ACCUMULATION IN CANINE VENTRICULAR MYOCYTES

Cyclic AMP regulation by muscarinic and adenosine receptors was investigated in isolated canine ventricular myocytes. Both the muscarinic receptor agonist, carbachol, and the adenosine receptor agonist, phenylisopropyladenosine, decreased isoproterenol-stimulated cyclic AMP accumulation in a concentration-dependent manner. Carbachol was more potent than phenylisopropyladenosine and had a greater inhibitory effect. At 10(-6) M, carbachol reduced isoproterenol-stimulated cyclic AMP by 73 +/- 5% while 10(-3) M phenylisopropyladenosine was required to decrease cyclic AMP accumulation by 54 +/- 8%. Pretreatment of myocytes with pertussis toxin to inactivate the inhibitory guanine nucleotide binding protein, Gi, completely abolished the effect of phenylisopropyladenosine to reduce cyclic AMP stimulation. In comparison, pertussis toxin treatment blunted the response to carbachol and shifted the dose-effect curve to the right but did not eliminate the inhibitory action of carbachol. In toxin-treated myocytes, 10(-3) M carbachol produced a 26 +/- 6% reduction of isoproterenol-induced cyclic AMP accumulation. This pertussis toxin-insensitive action of carbachol was antagonized by atropine and pirenzepine and was prevented when either of two different phosphodiesterase inhibitors. RO-20-1724 or isobutylmethylxanthine, was included in the incubation medium. The results indicate that adenosine receptor-mediated inhibition of hormone-stimulated cyclic AMP accumulation in ventricular myocytes occurs by a single, Gi-dependent mechanism while muscarinic inhibition appears to involve both Gi-dependent and Gi-independent mechanisms. The Gi-independent mechanism may reflect enhanced phosphodiesterase activity which results from the activation of muscarinic receptors.

Expression of the kallikrein/kinin system in human anterior segment

Tissue kallikrein acts on the substrate, low molecular weight kininogen, to liberate bradykinin in a variety of tissues. Bradykinin stimulation of B(2) kinin receptors has been shown to initiate signaling in trabecular meshwork cells and increase conventional outflow facility. The objective of the present study was to determine if the components for kinin generation and response are expressed in tissues of the human anterior segment. Expression of mRNA encoding tissue kallikrein (KK), low molecular weight kininogen, and B(1) and B(2) kinin receptors was examined in human ciliary smooth muscle (CM), trabecular meshwork (TM) and non-pigmented epithelial (NPE) cells using RT-PCR. Expression of component proteins was also investigated by immunohistochemical analyses performed on parasagittal sections of human anterior segment and TM cells, and by immunoblot. KK mRNA was detected in NPE cells and in cultured CM and TM cells from multiple donors. Each cell type also expressed mRNAs encoding both B(1) and B(2) kinin receptors. Immunohistochemical analysis of KK protein in sectioned anterior segment supported the RT-PCR results. Intense KK immunofluorescence was observed in the epithelial lining of the ciliary body and KK protein was also detected in the ciliary muscle. KK protein expression within the TM was demonstrated by analyses of TM tissue and cultured TM cells. The presence of KK along with B(1) and B(2) receptor proteins was confirmed by immunoblots of cell lysates prepared from CM, NPE or TM cells. Finally, both CM and TM cells were found to possess enzymes for bradykinin inactivation. These data demonstrate that key components for kinin generation and regulation are localized within the human anterior segment. Further, multiple cell types express both B(1) and B(2) kinin receptors and are targets for kinin action. The results support the possibility that kinins produced within the eye may contribute to the regulation of aqueous outflow.

Bradykinin activation of extracellular signal-regulated kinases in human trabecular meshwork cells.

Bradykinin stimulation of B(2) kinin receptors has been shown to promote matrix metallo-proteinase (MMP) secretion from trabecular meshwork cells and to increase conventional outflow facility. Because acute secretion of MMPs can be dependent on the activity of extracellular signal-regulated MAP kinases (ERK1/2), experiments were performed to determine bradykinin effects on ERK1/2 in cultured human trabecular meshwork cells and the relationship of these effects to MMP-9 release. Treatment of cells with bradykinin produced a rapid 4-to 6-fold increase in ERK1/2 phosphorylation. Stimulation of ERK1/2 activity peaked within 2 min and then declined to control levels by 60 min. The response maximum occurred with 100nM bradykinin and the estimated EC₅₀ was 0.7nM. Treatment of cells with the B₂ kinin receptor agonist, Tyr⁸- bradykinin, also stimulated ERK1/2 phosphorylation while the B₁ agonist, Lys- [Des-Arg⁹]- bradykinin had no significant effect. In addition, activation of ERK1/2 by bradykinin or Tyr⁸- bradykinin was blocked by the selective B₂ receptor antagonist, Hoe-140. Inhibition of MAP kinase kinase (MEK) with U0126 also blocked bradykinin-induced ERK1/2 phosphorylation. Suppression of protein kinase C activity with the nonselective inhibitor, GF109203X, or by down-regulation with phorbol ester, diminished, but did not eliminate, bradykinin activation of ERK1/2. A similar decrease of ERK1/2 stimulation was observed when Src kinase was inhibited by 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Finally, blockade of bradykinin-induced ERK1/2 activation substantially reduced the peptides action to stimulate MMP-9 release into the extracellular environment. The data demonstrate that bradykinin promotes ERK1/2 activation in human trabecular meshwork cells. The effect is mediated by B₂ kinin receptors, involves two different signaling pathways, and results in increased secretion of MMP-9.

Myocardial Sensitivity to Isoproterenol Following Abrupt Propranolol Withdrawal in Conscious Dogs

Summary The sensitivity of the cardiovascular system to isoproterenol following abrupt cessation of propranolol administration was studied in chronically instrumented conscious dogs pretreated orally with propranolol for 14 days. The effects of intravenous isoproterenol on heart rate, blood pressure, and mean velocity of circumferential fiber shortening (Vef) were determined before and during propranolol administration and then at 12 hr intervals following drug withdrawal. During propranolol administration, all dose-response curves to isoproterenol were predictably shifted to the right. Twenty-four hours following propranolol withdrawal, when plasma levels of drug averaged only 7 ng/ml, the chronotropic response to isoproterenol remained significantly inhibited. In contrast, the Vef response recovered rapidly and at 24 hr equaled the pretreatment control response. Furthermore, between 36 and 60 hr, when plasma propranolol levels averaged less than 1 ng/ml, the Vef dose-response curve to isoproterenol was shifted to the left of control prior to returning to the pretreatment level. This leftward displacement of the Vef response curve was not accompanied by a parallel change in the heart rate response or in afterload. Plasma norepinephrine levels were measured throughout the study and were not altered during drug treatment or following withdrawal. Thus, the data indicate that abrupt cessation of propranolol administration is associated with a period of increased myocardial sensitivity to at least the inotropic effects of isoproterenol and that abrupt withdrawal does not result in acute changes in sympathetic nervous system activity.