N-[2-hydroxy-5-[2-(methylamino)ethyl]phenyl]methanesulfonamide. A potent agonist which releases intracellular calcium by activation of alpha 1-adrenoceptors.

N-[2-Hydroxy-5-[2-(methylamino)ethyl]phenyl]methanesulfonamide (SK&F 102652) has been prepared and characterized pharmacologically. It is a potent agonist with an EC50 of 25 nM at alpha 1-adrenoceptors as determined in the isolated perfused rabbit ear artery. On the presynaptic alpha 2-adrenoceptors of the guinea pig atrium it was considerably weaker, demonstrating an EC50 for inhibition of neurotransmission of 1200 nM and thus an overall alpha 1/alpha 2 selectivity ratio of greater than or equal to 48. In the vascular smooth muscle of the canine saphenous vein an EC100 concentration of this agonist in the presence of zero external Ca2+ induced 37.9 +/- 1.4% of the maximal contractile response due to this agent while the endogenous ligand norepinephrine evoked only 14.5 +/- 0.4% of the maximum. In the presence of low (1 microM) external calcium, this agent produced 78.3 +/- 5.3% of maximum while norepinephrine gave 45.3 +/- 7.4%. This agent produces alpha 1-adrenoceptor-mediated contraction primarily by release of intracellular Ca2+ and should provide a useful tool for characterizing alpha 1-receptor subtypes.

Development of an affinity ligand for purification of alpha 2-adrenoceptors from human platelet membranes.

Human platelets contain alpha 2-adrenoceptors which are negatively coupled to the enzyme adenylate cyclase. In order to better understand the interaction of this subtype of alpha receptor with this key enzyme, we have initiated a program to isolate and characterize the alpha 2-adrenoceptor. This report describes the synthesis and biological characterization of a series of molecules that were prepared as affinity ligands for this purpose. The best of these is 9-(allyloxy)-6-chloro-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SK&F 101253). This compound is an alpha 2-adrenoceptor antagonist, which was obtained by synthetic modification of 6-chloro-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SK&F 86466), a novel antagonist with high affinity for the alpha 2-receptor.

Evidence that oxmetidine inhibits transmembrane-calcium flux in cardiac and vascular tissue.

Oxmetidine, at concentrations in excess of 1 X 10(-6)M, caused concentration-dependent negative inotropic and chronotropic responses in guinea-pig isolated heart preparations. Oxmetidine, at concentrations in excess of 1 X 10(-5)M, caused negative inotropic responses in guinea-pig papillary muscle preparations. The negative inotropic responses to oxmetidine were associated with shortening of the plateau phase of the action potential. Verapamil and nifedipine caused similar shortening of the plateau phase of the action potential at equivalent negative inotropic concentrations indicating that oxmetidine may also act as a calcium antagonist. In preparations partially depolarized by raising extracellular K+ concentration, oxmetidine also exhibited negative inotropic activity and reduced the calcium-dependent action potential. However, unlike verapamil and nifedipine, oxmetidine did not show voltage-dependent activity. Oxmetidine, at concentrations in excess of 1 X 10(-5)M, inhibited Ca2+-dependent contractions of dog saphenous vein preparations and inhibited 45Ca2+-uptake into veins depolarized by high extracellular K+. In vivo, these calcium antagonist actions of oxmetidine were demonstrated by vasodilatation, reduction in blood pressure, bradycardia and reduced cardiac output in anaesthetized cats. Oxmetidine, at concentrations of 1 X 10(-5)M and above, shows properties consistent with inhibition of transmembrane Ca2+ flux. This action can be distinguished from other calcium antagonists as the effects of oxmetidine are not voltage-dependent.

Suppression of pentylenetetrazol-elicited seizure activity by intraosseous propranolol in pigs.

The intraosseous (IO) route provides a rapid and effective alternative venous access in the pediatric population when the conventional intravenous (IV) route cannot be easily obtained. DL-propranolol, a beta-adrenoceptor antagonist, exhibits antiepileptic activity in various animal seizure models. This study assessed the efficacy of IO propranolol in suppressing pentylenetetrazol (PTZ)-induced seizure activity in pigs. Domestic swine (13-20 kg) were prepared for recordings of arterial blood pressure, ECG and electrocortical activity. Seizure activity was induced by pentylenetetrazol (PTZ; 100 mg/kg; IV). Sixty seconds after the onset of seizure activity, the animals either received no drug (control) or propranolol (IV or IO via an 18-gauge spinal needle placed in the right proximal tibia). A transient increase (16.3-50.0%) in the mean arterial blood pressure (MAP) was observed following PTZ administration. Both IO and IV propranolol significantly suppressed the seizure duration (SD) (sec/min interval) at 1 min following drug administration; SD control, 36.3 +/- 4.8; IV propranolol, 12.3 +/- 5.1; IO propranolol, 18.3 +/- 6.0. In addition, both IV and IO propranolol produced a maximal decrease of 32-38% in the basal heart rate; and reduced the transient increase in MAP elicited by PTZ, with no significant effect on the basal MAP. The data demonstrate that 1) propranolol possesses anticonvulsant activity against PTZ-induced seizure in the pig, and 2) the intraosseous route is a rapid and effective alternative venous access for propranolol administration in swine.

An investigation of the pathological and physiological effects of intraosseous sodium bicarbonate in pigs.

Recent interest in the intraosseous (IO) route as an alternative venous access for drug and fluid administration has increased. This study examined the physiological and skeletal pathological effects of IO NaHCO3 in pigs. In the pathological studies, swine (8-10 kg) received NaHCO3 (1 mEq/kg) in one tibia and saline (1 ml/kg) in the other tibia via an 18-gauge spinal needle inserted into the anteromedial surface of the bone. The animals were then observed for one month, sacrificed, and the tibias were isolated, sectioned, and stained for pathological examinations. The physiological effects of IO NaHCO3 infusion were studied and compared with that of intravenous (IV) administration using a cardiac arrest model as previously described. The results demonstrated that NaHCO3 had no effect on the mean arterial blood pressure and plasma catecholamine levels, but increased arterial pH values within two minutes of administration. Similar effects were found with IV NaHCO3. Pathological data indicated signs of minimal local increase in skeletal turnover associated with IO NaHCO3 infusion. It is concluded that the IO route is a safe alternative venous access for NaHCO3 administration in swine.

Measurements of 45Ca2+ uptake and contractile responses after activation of postsynaptic alpha 1-adrenoceptors in the isolated canine saphenous vein: effects of calcium entry blockade.

The increase in 45Ca2+ content produced by the EC100 concentrations of a series of alpha 1-agonists in the canine saphenous vein (CSV) was determined in the presence of 10(-7) M rauwolscine and found to be correlated (r = 0.92) with the intrinsic activities of the alpha 1-agonists in CSV. The degree of inhibition of 45Ca2+ uptake by nifedipine (1 microM) was inversely correlated (r = -0.97) with the intrinsic activities of the alpha 1-agonists and also inversely correlated (r = -0.95) with the 45Ca2+ uptake induced by the agonists. In the presence of 5 mM LaCl3, there was no significant 45Ca2+ uptake elicited by KCl (80 mM) or activation of postsynaptic alpha 1-adrenoceptors in this tissue, and the alpha 1-agonists were found to have a varied ability to release internal Ca2+ for contractions. It is concluded that (1) activation of alpha 1-adrenoceptors in CSV utilizes both intracellular and extracellular Ca2+ for contractions; (2) the increase in 45Ca2+ content after activation of post-synaptic alpha 1-adrenoceptors in CSV is directly correlated with the intrinsic ability of the alpha 1-agonists to induce contractions; and (3) the sensitivity of the 45Ca2+ uptake to nifedipine is inversely related to the intrinsic ability of the alpha 1-agonists to translocate extracellular Ca2+.

Correlation of [14C]muscimol concentration in rat brain with anticonvulsant activity.

Muscimol, an in vivo and in vitro GABA agonist, has anticonvulsant activity against bicuculline-induced seizures when given systemically to rats. To determine whether parent compound or a metabolite possessed the anticonvulsant activity, experiments were performed with [14C]muscimol. Anticonvulsant activity was determined by the percent of animals protected against tonic forelimb extension induced by bicuculline. Brain and urine were analyzed for unchanged [14C]muscimol by thin-layer chromatography. The time course of anticonvulsant activity and [14C]muscimol concentration in brain after intravenous injection were similar. Peak brain concentration of [14C]muscimol and maximal protection against bicuculline-induced seizures occurred simultaneously. These data suggest that intravenously administered [14C]muscimol rapidly penetrates brain tissue and parent compound is responsible for antagonism of bicuculline-induced convulsions.

Alpha 1-adrenoceptor-mediated vasoconstriction in vivo to enantiomers of SK & F 89748-A.

The pressor activity of the 1-enantiomer of SK & F 89748-A, 1,2,3,4-tetrahydro-8-methoxy-5-(methylthio)-2-naphthalenamine, in pithed normotensive rats was found comparable with that of 1-phenylephrine. The d-enantiomer was half as potent. The log dose-pressor effect curves for d- and 1-SK & F 89748-A were not influenced by reserpine treatment (2 X 5 mg/kg i.p., -48 and -24 h), were virtually unaffected by yohimbine (1 mg/kg i.v., -15 min) but were markedly shifted to the right by prazosin (0.1 mg/kg i.v., -15 min) and phentolamine (1 mg/kg i.v., -15 min). Similar observations were made for the 1-enantiomer in pithed cats. It is concluded that d- and 1-SK & F 89748-A are potent, directly acting highly selective agonists of (vascular) postjunctional alpha 1-adrenoceptors. Potency and selectivity were equally pronounced for both enantiomers. The currently available selective agonists of alpha 1-adrenoceptors, including the optical isomers of SK & F 89748-A, cannot distinguish between alpha 1- and alpha 2-adrenoceptors. This conclusion is based on binding affinity since these affinities are linearly correlated as shown by radioligand displacement experiments.

Assessment of alpha-adrenergic receptor subtypes in isolated rat aortic segments.

The postsynaptic alpha-adrenoceptors in the isolated rat aorta have been characterized according to the sensitivity of the tissue to selective alpha 1- and alpha 2-adrenoceptor agonists and antagonists. The potency (-log EC50) order of the non-selective alpha-agonist norepinephrine and relatively selective agonists was as follows: norepinephrine (alpha 1 = alpha 2; 7.30); clonidine (alpha 2 greater than alpha 1; 7.01); phenylephrine (alpha 1 greater than alpha 2; 6.99), SK & F 89748--A (alpha 1 greater than alpha 2; 6.65); BHT-920 (alpha 2 much greater than alpha 1; 5.56) and M-7 (alpha 2 greater than alpha 1; 4.66). The isolated rat aorta was 12-200-fold more sensitive to the alpha 1-adrenoceptor agonists phenylephrine and SK & F 89748-A, than to the alpha 2-agonists, BHT-920 and M-7. Prazosin is 245-1259-fold more potent than rauwolscine as an antagonist of contractions induced by various alpha 1- and alpha 2-agonists in the rat aorta. These data indicate that constriction of the smooth muscle of the rat aorta to alpha-adrenergic agonists is mediated through alpha 1- but not alpha 2-adrenoceptors.

Evidence for and against heterogeneity of alpha 1-adrenoceptors.

Recent experimental evidence has suggested that the alpha 1 adrenoceptor may need to be further subdivided. It can no longer be stated categorically that alpha 1-adrenoceptors are present only at postjunctional sites, in view of several reports of alpha 1-mediated modulation of adrenergic and cholinergic neurotransmission. Furthermore, comparison of the pharmacologic characteristics of the alpha 1-adrenoceptor in different species and/or tissues can show clear differences in sensitivity to selective agonists and antagonists, and differences in the degree of dependence on extracellular calcium. However, in other cases, alpha 1-adrenoceptors at diverse sites have been found to have identical characteristics. Furthermore, the subcategories identified by the various selective agents do not fall into the same discrete groups, in contrast to division of alpha-adrenoceptors into alpha 1 and alpha 2-adrenoceptors. Therefore, at this time it seems premature to subdivide the alpha 1-adrenoceptor further.

An investigation of the cardiotoxic action of vancomycin in the isolated working rat heart.

The mechanism of cardiotoxic action of vancomycin was examined in preparations of isolated working rat hearts and spontaneously beating right atria. Vancomycin produced a concentration-dependent decrease in aortic flow, coronary flow and heart rate in the isolated working rat heart, with no significant effect on other haemodynamic parameters. At 5 mm, vancomycin produced a statistically significant decrease (compared with control values) in aortic flow (24.1 +/- 7.5%) and in the basal heart rate (34.3 +/- 3.5%) after 15 min incubation. Coronary flow was also reduced by 23.5 +/- 9.2%. Prolonged exposure of the preparation to 5 mm-vancomycin produced a marked time-dependent bradycardia accompanied by a time-dependent increase in the leakage of lactic dehydrogenase (LDH) into the perfusion medium. Moreover, a correlation (r = -0.94) was found between the time-dependent bradycardia and LDH leakage induced by Vancomycin. In the isolated spontaneously beating right atria pretreated with atropine, vancomycin (5 mm) also produced a time-dependent bradycardia similar to that found in the isolated working rat heart. Moreover, (45)Ca(2+)-flux studies indicated that vancomycin had no significant effect on the (45)Ca(2+) uptake into the right atrial muscle. These data suggest that: (1) vancomycin has a direct and acute cardiotoxic action at high concentrations (> 1 mm); (2) time-dependent bradycardia is a sensitive functional index for the cardiotoxicity induced by vancomycin; (3) the bradycardia elicited by vancomycin is due neither to the release of acetylcholine from the parasympathetic nerves innervating the heart nor to blockade of Ca(2+) entry.

The ocular hypotensive action of SK&F 86466 in the conscious rabbit.

SK&F 86466, 6-chloro-3-methyl-2, 3, 4, 5-tetrahydro-1H-3-benzazepine, a potent and selective competitive antagonist of the alpha 2-adrenoceptor is of interest as a potential antiglaucoma agent. Following instillation into the rabbit eye, SK&F 86466 produced concentration (0.25 to 10%) dependent reductions in intraocular pressure (IOP). The ocular hypotensive effect of SK&F 86466 was maximal 30-60 minutes post treatment and persisted for at least 90 minutes. The IOP of the contralateral (untreated) eye was significantly decreased only at the 10% concentration. A 10% solution of SK&F 86466 had no significant effect on mean arterial blood pressure (MAP) of rabbits during the time of maximal bilateral ocular hypotensive activity. These results suggest: (1) a selective alpha 2-adrenoceptor antagonist can reduce IOP when applied topically to rabbit eyes and (2) the ocular hypotensive effect of SK&F 86466 is not secondary to a reduction in systemic blood pressure.

Role of extracellular calcium in contractions produced by activation of postsynaptic alpha-2 adrenoceptors in the canine saphenous vein.

Canine saphenous vein (CSV) has been shown to contain both postsynaptic alpha-1 and alpha-2 adrenoceptors. Our previous studies have shown that activation of postsynaptic alpha-1 adrenoceptors in this tissue utilizes both extracellular and intracellular Ca++ to produce contractions. In the present study, the source of calcium mobilized by activation of postsynaptic alpha-2 adrenoceptors in CSV was elucidated. Contractions of tissue rings to the supramaximal concentrations of three selective alpha-2 agonists, B-HT 920, M-7 and clonidine, were determined in the absence and presence of 5 mM La . In the presence of La , clonidine and M-7 produced small but statistically significant contractions (8-14% of control) which were abolished when the alpha-1 adrenoceptors were inactivated by phenoxybenzamine (10(-7) M, 30 min). In contrast, contractions to B-HT 920 were abolished completely in the presence of La . All the three alpha-2 agonists stimulated 45Ca++ uptake into CSV (0.3-0.4 mmol/kg wet weight, 10 min). 45Ca++ efflux studies demonstrated that the selective alpha-2 agonist, B-HT 920 (10(-5) M plus 10(-7) M phenoxybenzamine), did not induce an increase in the rate of 45Ca++ efflux. In contrast, an augmented 45Ca++ efflux rate was observed with the alpha-1 agonist, phenylephrine (10(-4) M plus 10(-7) M rauwolscine). These results suggest that activation of postsynaptic alpha-2 adrenoceptors in CSV utilizes primarily extracellular Ca++ to produce contractions.

Comparison of the calcium antagonistic activity of nifedipine and FR34235 in the canine saphenous vein.

The effects of FR34235 (FR) on contractions and 45Ca2+ uptake stimulated by depolarization (80 mM KCl) and activation of postsynaptic alpha 1-adrenoceptors in the canine saphenous vein (CSV) were studied and compared with those of nifedipine. The 45Ca2+ uptake and contractions evoked by KCl were inhibited in a dose-dependent fashion by FR [concentration producing 50% inhibition [(IC50 = 2.0 +/- 0.5 and 1.2 +/- 0.3 X 10(-8) M, respectively)] and nifedipine (IC50 = 6.0 +/- 1.0 and 4.0 +/- 0.9 X 10(-8) M, respectively). FR was a potent inhibitor of the 45Ca2+ uptake and contractions of CSV induced by the selective alpha 1-agonist, SK&F l-89748 (l-[1,2,3,4-tetrahydro-8-methoxy-5-(methylthio)-2-naphthalenamine] ) (IC50 = 7.2 +/- 0.7 and 7.0 +/- 1.8 X 10(-8) M, respectively). In contrast, the contractions and 45Ca2+ uptake elicited by SK&F l-89748 were much less sensitive to nifedipine (IC50 = 6.0 +/- 1.4 and 6.6 +/- 0.4 X 10(-6) M, respectively). The results suggest the following: (a) both FR and nifedipine are potent antagonists of the voltage-dependent Ca2+ channels in CSV; and (b) FR is a more effective antagonist of receptor-operated Ca2+ entry in the saphenous vein than nifedipine.

Effects of chemical sympathectomy by 6-hydroxydopamine on alpha-adrenoceptor-mediated pressor responses in pithed rat.

The effects of 6-hydroxydopamine (6-OHDA) treatment of neonatal and adult rats on vasopressor responses to postsynaptic alpha-adrenoceptor activation were studied in adult pithed rats. The pressor response to electrical stimulation of the thoracosympathetic outflow (T7-T9) was markedly reduced after neonatal or adult 6-OHDA treatment, indicating a functional destruction of the sympathetic input to the vasculature. Pressor response curves to injected noradrenaline (a nonselective alpha-agonist) and phenylephrine (a selective alpha 1-agonist) were shifted to the left in all 6-OHDA-treated animals. However, the pressor response to the selective alpha 2-agonist M-7 (5,6-hydroxy-2-dimethylamino tetralin) was unaltered in all the 6-OHDA-treated animals. It is concluded that functional destruction of peripheral sympathetic nerve endings in vascular smooth muscle by treatment of neonatal or adult rats with 6-OHDA produces an increased pressor response to alpha 1-adrenoceptor agonists, but not to alpha 2-adrenoceptor agonists in the pithed rat.