Peter W. Abel

β1- andβ2-adrenoceptor binding and functional response in right and left atria of rat heart

SummaryThe properties ofβ1- andβ2-adrenoceptors in right and left atria of rat heart, and their roles in mediating chronotropic and inotropic responses toβ-adrenoceptor agonists were examined. [125I](-)-pindolol (125IPIN) bound saturably and specifically to a single class of high affinity sites in homogenates of both right and left atria. Thek1s for association in right and left atria were 6.5×109 l/mol-min and 2.3×109 l/mol-min respectively, while thek−1s for dissociation were 0.20 min−1 and 0.17 min−1. The kinetically determinedKDs were 75 pmol/l in right and 30 pmol/l in left atria and were similar to the equilibriumKDs determined from Scatchard analysis of saturation isotherms of specific125IPIN binding. Inhibition of125IPIN binding byβ-adrenoceptor antagonists was stereoselective and the order of potency was timolol > 1-propranolol > d-propranolol > sotalol. Inhibition byβ1- andβ2-adrenoceptor subtype selective antagonists yielded flat displacement curves with low Hill coefficients. Nonlinear regression analysis of displacement byβ1-selective (practolol, atenolol and metoprolol) andβ2-selective (ICI 118,551) antagonists gave estimates of the proportion ofβ1- andβ2-adrenoceptors present in rat atria. Right atria contained 67±4.2%β2-adrenoceptors and 33±4.2%β2-adrenoceptor, while left atria contained 67±2.8%β1- and 33±2.8%β2-adrenoceptors. Increases in the rate of spontaneously beating right atria and the force of electrically driven left atria caused byβ-adrenoceptor agonists were also measured. pA2 values for non-subtype selectiveβ-adrenoceptor antagonists in inhibiting isoprenaline-induced increases in rate and force were highly correlated withKD values determined for specific125IPIN binding. pA2 values forβ1- andβ2-selective antagonists in inhibiting isoprenaline-induced increases in rate and force correlated well with the pKD values of these drugs in binding toβ1-adrenoceptors, but not with the pKD values in binding toβ2-adrenoceptors. Dose-response curves for stimulation of both rate and force by theβ2-selective agonists procaterol and zinterol were shifted to a much greater extent by selective blockade ofβ1-adrenoceptors with metoprolol than by selective blockade ofβ2-adrenoceptors with ICI 118,551, suggesting that these compounds caused their effects by activatingβ1-adrenoceptors. These results suggest thatβ1- andβ2-adrenoceptors coexist in both left and right atria of rat heart in approximately a 2∶1 ratio, however onlyβ1-adrenoceptors mediate the chronotropic and inotropic effects ofβ-adrenoceptor agonists.

Neuropeptide Y potentiates contraction and inhibits relaxation of rabbit coronary arteries

The effects of neuropeptide Y (NPY) on contraction and relaxation of isolated rabbit coronary arteries were studied. NPY alone caused a weak contraction of coronary arteries with a mean EC50 value of 29 ± 2.0 nM. Following exposure of coronary arteries to 30 nM NPY, the potencies of norepinephrine (in the presence of 3 μM timolol) and histamine in causing contraction were increased twofold, with no change in maximal contraction. After half-maximal contraction of coronary arteries with histamine and addition of 30 nM NPY, relaxation produced by norepinephrine (in the presence of 3 μM phentolamine), adenosine, and acetylcholine was inhibited. Concentration-response curves for all vasodilators were shifted to the right 10–22-fold by 30 nM NPY. Maximal relaxation caused by adenosine and norepinephrine was not changed by NPY, whereas the maximal response to acetylcholine was 37% less in the presence of NPY. Correlation of the tension produced by NPY with the shift in agonist contraction or relaxation concentration–response curves indicated that NPY-induced in-creases in baseline tone had no effect on the degree of shift in agonist concentration–response curves. These results show that NPY causes a modest potentiation of agonist-induced contraction and a dramatic blockade of vasodilator-induced relaxation of rabbit coronary arteries.

Activation of α1-adrenoceptors increases [3H]inositol metabolism in rat vas deferens and caudal artery

Rings of rat vas deferens and caudal artery were incubated with [3H]inositol in Krebs-Ringer bicarbonate buffer containing 10 mM lithium chloride, and the production of water-soluble [3H]inositol phosphates was monitored. Norepinephrine increased [3H]inositol phosphate accumulation 7-fold in rings from vas deferens and 3-fold in rings from caudal artery. Epinephrine, phenylephrine and methoxamine were as effective as norepinephrine, suggesting that these drugs are full agonists in causing this response. Prazosin, phentolamine and yohimbine completely blocked the stimulation by norepinephrine in both tissues with potencies typical of blockade of alpha 1-adrenoceptors. Despite a substantial receptor reserve for alpha 1-adrenoceptor mediated contractile responses, clonidine, p-amino-clonidine, phenylpropanolamine and ephedrine can only cause a partial contractile response in rat vas deferens. However, all of these partial agonists were either as effective or more effective in increasing [3H]inositol phosphate accumulation in rat vas deferens as they were in activating a contractile response. These data suggest that alpha 1-adrenoceptors increase phosphatidylinositol turnover in rat vas deferens and caudal artery, and that there may be a receptor reserve for alpha 1-adrenoceptor mediated increases in [3H]inositol phosphate accumulation in these smooth muscles.

High levels of NPY in rabbit cerebrospinal fluid and immunohistochemical analysis of possible sources

We have analyzed rabbit cerebrospinal fluid for neuropeptide Y (NPY)-like immunoreactivity, using high performance liquid chromatography (HPLC) and radioimmunoassay (RIA) and examined the anatomical relationship of NPY-containing fibers to the cerebral vasculature and the third cerebral ventricle. Cerebrospinal fluid (CSF) obtained from the cisterna magna of rabbits was injected into a C18 column and subjected to HPLC. The fractions were collected, dried and reconstituted in buffer for NPY radioimmunoassay. A single peak of NPY immunoreactivity was obtained which corresponded in retention time to synthetic porcine NPY. Analysis of CSF samples produced displacement curves parallel to the standard curve. Immunohistochemistry revealed numerous NPY-labeled fibers which penetrated the ependymal lining of the third cerebral ventricle and directly bordered the ventricular lumen. Other fibers were observed in the pia which lines the ventral aspect of the hypothalamus. The basilar artery, its branches and other cerebral vessels were surrounded by NPY-labeled fibers. The results show that: (1) approximately 1 ng/ml of NPY immunoreactivity which corresponds chromatographically to synthetic porcine NPY is present in rabbit CSF; (2) NPY-containing fibers surround the basilar artery and other cerebral vessels; (3) NPY may be released into the CSF from axons in the pia and from axons which penetrate the ependymal lining of the third ventricle. These observations form the basis for our analysis of the vasoconstrictor effects of NPY and its role in cerebrovasospasm after experimental subarachnoid hemorrhage.

Neuropeptide Y: vasoconstrictor effects and possible role in cerebral vasospasm after experimental subarachnoid hemorrhage

The possible role of neuropeptide Y (NPY) as a vasoconstrictor substance contributing to the development of cerebral vasospasm after experimental subarachnoid hemorrhage was studied. Autologous blood was injected into the cisterna magna of control rabbits and rabbits that had received bilateral superior cervical ganglionectomy 2 days before blood injection. Three days after blood injection the concentration of NPY in cerebrospinal fluid (CSF) was 5971 +/- 551 pg/ml while CSF from control animals contained 992 +/- 162 pg/ml of NPY. The effects of porcine NPY on norepinephrine-induced contraction of rabbit cerebral arteries were also studied in vitro. NPY (1.2 nM) caused a 3-fold potentiation of norepinephrine-induced contraction of cerebral arteries. CSF from control rabbits diluted by 50% with Krebs solution had no significant effect on norepinephrine-induced contraction of cerebral arteries when compared to responses in Krebs solution only; however, diluted CSF from denervated blood-injected rabbits potentiated norepinephrine-induced contraction by 2.6-fold. Antiserum containing NPY specific antibodies was used to immunoprecipitate the peptide from CSF taken from denervated blood-injected rabbits. CSF treated with this antiserum contained less than 40 pg/ml of NPY and had no effect on norepinephrine-induced contraction of cerebral arteries. These results show that the concentration of NPY in CSF of rabbits is elevated after experimental subarachnoid hemorrhage. In addition, NPY in CSF can potentiate the vasoconstrictor effect of norepinephrine which may contribute to the development of cerebral vasospasm.

Relationship between α1-adrenoreceptor density and functional response of rat vas deferens

SummaryThe relationship between the density of α1-adrenoreceptors and the longitudinal contractile response of rat vas deferens was examined by using phenoxybenzamine to irreversibly decrease α1-adrenoreceptor density. Receptor density was measured by Scatchard plots of saturation analysis of specific 125I-BE 2254 (125IBE) binding and compared to the potency of α-adrenoceptor agonists in causing contraction and the maximum contraction elicited by these agonists. Treatment of isolated vasa deferentia in organ baths with phenoxybenzamine caused a dose-dependent decrease in the density of 125IBE binding sites, the potency of the full agonist phenylephrine in activating contraction, and the maximum contractile response. The percentage of functional receptors remaining after phenoxybenzamine treatment (q value) was calculated from the contraction data and compared to the percentage of 125IBE binding sites remaining. The reduction in the number of functional receptors was much greater than the reduction in the number of 125IBE binding sites at all doses of phenoxybenzamine. Since the magnitude of the contractile response caused by a weak partial agonist should be approximately proportional to the density of functional receptors in the tissue, partial agonists were used as an independent measure of the degree of functional receptor inactivation. Bath application of two different doses of phenoxybenzamine caused a decrease in the maximal contraction caused by the partial agonists clonidine and ephedrine which was similar to the calculated decrease in the q value, but not to the observed decrease in the density of 125IBE binding sites. Phenylephrine also caused contraction of spiral strips prepared from vas deferens, suggesting that α1-adrenoreceptors exist on the inner circular muscle as well as the outer longitudinal muscle of this tissue. To determine whether access barriers in the layered muscle structure of the vas deferens could result in a preferential inactivation of a particular subpopulation of receptors, phenoxybenzamine was applied more uniformly by intravenous administration to anesthetized rats. Intravenous administration of phenoxybenzamine also resulted in a dose-dependent decrease in the density of 125IBE binding sites, the potency of phenylephrine in activating contraction, and the maximum contractile response. The degree of functional receptor inactivation after intravenous phenoxybenzamine administration (q values) agreed reasonably well with the observed reduction in 125IBE binding sites, particularly at lower doses of phenoxybenzamine. The equilibrium constant for activation of the receptor by phenylephrine (Kact) was the same whether calculated using data from bath application of phenoxybenzamine or intravenous application of phenoxybenzamine. These data suggest that only a portion of the α1-adrenoreceptors in rat vas deferens affect longitudinal contraction. There is, however, a large pool of spare receptors for longitudinal contraction and these receptors are preferentially inactivated by application of phenoxybenzamine to the isolated tissue in an organ bath. When phenoxybenzamine is applied intravenously, the receptors affecting longitudinal contraction are inactivated to a degree similar to that of the total pool of receptors in this tissue.

Binding of agonists and antagonists to β-adrenoceptors in rat vas deferens: relationship to functional response

SummaryThe properties of β-adrenoceptors in rat vas deferens were examined using radioligand binding assays of 125I-pindolol (125IPIN) and inhibition of electrically-evoked contractions of vas deferens in vitro. 125IPIN labelled a single class of high affinity binding sites with apparently mass action kinetics in membrane preparations of vas deferens with properties consistent with an essentially homogeneous population of β2-adrenoceptors. Isoprenaline inhibited electrically evoked (60 V, 1.0 ms, 0.1 Hz) contractions of vas deferens with an EC50 of 18.0±2.1 nM. KB values for antagonists in competitively antagonizing this response correlated well (r2=0.99) with the KD values for inhibition of 125IPIN binding. Inhibition of 125IPIN binding by isoprenaline, adrenaline, noradrenaline and salbutamol was determined under conditions designed to produce high and low affinity agonist binding. In the presence of 10 mM MgCl2, agonists inhibited specific 125IPIN binding with a relatively high potency and low Hill slope, while in the presence of 154 mM NaCl and 300 μM guanosine-5′-triphosphate, agonists inhibited specific 125IPIN binding with a lower potency and an apparent Hill slope closer to 1. To determine which affinity state was relevant to functional receptor stimulation, receptor density was decreased with bromoacetylalprenololmenthane (BAAM). Treatment of membrane preparations with 0.3 μM BAAM produced a 45% decrease in the Bmax for 125IPIN with no change in the apparent KD. Treatment of intact vasa deferentia with increasing concentrations of BAAM resulted in a progressive rightward shift in the dose-response curve to isoprenaline or salbutamol folowed by a decreased maximum response. KA values for isoprenaline and salbutamol in activating the functional β-adrenoceptors were compared with KI values for agonist inhibition of specific 125IPIN binding. The KA values for both agonists were not significantly different from the low affinity KI values, but were significantly different from the high affinity KI values. These data suggest that 1) a homogeneous population of β2-adrenoceptors inhibiting contraction of rat vas deferens can be labelled with 125IPIN, 2) there is a substantial β-adrenoceptor reserve in rat vas deferens; and 3) the initial event in signal transduction by β-adrenoceptors in rat vas deferens is the binding of agonists to the low affinity form of the receptor which is not complexed with the guanine nucleotide binding protein.

Vasodilator drug effects on internal mammary artery and saphenous vein grafts.

The internal mammary artery is a dynamic conduit used for myocardial revascularization in which potential exists for spasm as well as for vasodilation. This study investigated vasodilator drug effects on the mammary artery using nitroprusside and nitroglycerin in vitro to measure the inhibition of contraction of human internal mammary artery and in vivo to examine blood flow through a canine mammary artery. In the in vitro study, ring segments of human internal mammary arteries were suspended on strain gauges in muscle baths containing 37 degrees C Krebs solution for measurement of isometric tension in vitro. Arterial contraction was stimulated with 70 mM potassium and 10 microM norepinephrine, and inhibition of contraction by vasodilators was measured. Nitroprusside was more potent and effective than was nitroglycerin in inhibiting potassium and norepinephrine contraction. The in vivo study utilized a canine (n = 8) right heart bypass preparation that allowed precise control of cardiac output, blood pressure and heart rate, which were maintained constant. The internal mammary artery graft and the saphenous vein graft perfused the same coronary artery bed. Electromagnetic flow probes measured graft flow (with the other graft occluded) before and after 15 min of drug infusion (1 microgram/kg per min). Nitroglycerin significantly increased mammary artery flow 36 +/- 13%, whereas nitroprusside significantly decreased it 12 +/- 2%. Saphenous vein grafts responded differently; graft blood flow decreased with nitroglycerin and increased with nitroprusside. Thus, although nitroprusside was more effective than nitroglycerin in inhibiting mammary artery contraction in vitro, it decreased internal mammary artery graft flow measured in vivo. Nitroglycerin had the opposite effect, increasing mammary graft flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of β-adrenoceptor density and function in rat vas deferens

Abstract β-Adrenoceptor density and responsiveness were examined in rat vas deferens following surgical and pharmacological treatments. Receptor density was measured by Scatchard analysis of saturation isotherms of specific [ 125 I]pindolol ([ 125 I]PIN) binding in membrane homogenates. Functional responsiveness was measured by isoprenaline-induced inhibition of field stimulated (60 V, 1 ms, 0.1 Hz) or 40 mM K + -induced contractions. Four days following surgical denervation of vas deferens there was no change in the density of [ 125 I]PIN binding sites, suggesting that these sites are not located on prejunctional neurons. Neither 7 day bilateral adrenalectomy, 21 day denervation, nor 7 days treatment with 10 mg/kg per day desmethylimipramine caused changes in either the potency of isoprenaline in inhibiting contraction or the density of [ 125 I]PIN binding sites compared to controls. Infusion of 3 mg/kg per day isoprenaline for 8 days significantly reduced the potency of isoprenaline in inhibiting field stimulated contractions, reduced the maximum degree of inhibition, and reduced the density of [ 125 I]PIN binding sites. These results suggest that β-adrenoceptor density and responsiveness in rat vas deferens are not affected by removal of adrenal hormones or neuronal stimulation, but that receptor density and responsiveness can be decreased by increasing the concentration of β-adrenoceptor agonists at the receptor. Therefore, β-adrenoceptors in rat vas deferens probably receive little tonic stimulation under normal circumstances.

Relationship of alpha-1 Adrenergic Receptor Occupancy to Tissue Response

A curious aspect of receptor function is that the relationship between the occupancy of a receptor pool by an agonist drug and production of a tissue response very often is not linear. In many tissues a maximal tissue response can be evoked when only a very small fraction of the receptor pool is occupied by agonist (Stephenson, 1956; Nickerson, 1956). The remaining receptors are referred to as “spare” receptors or receptor “reserve.” Since the extent of the receptor reserve can vary dramatically from tissue to tissue for the same receptor type, there is no predictable relationship between the concentration of agonist necessary to occupy a particular fraction of the receptor pool and the concentration necessary to cause the same fractional tissue response.