Catecholamine and divalent cation effects on frog liver adenylate cyclase

Frog liver adenylate cyclase was characterized with respect to divalent cation interaction and hormonally stimulated activities. The enzyme catalyzed the synthesis of cyclic [³²P]3′,5′-AMP from α-³²P-labeled ATP. The activity of the enzyme was linear with time and00 protein concentration. The K_m for ATP was 0.5 mM, in the presence or absence of stimulators. The temperature optimum was 25°. GTP (10^?4M) increased the stimulation of adenylate cyclase by epinephrine. Similar activities were obtained using 5 mM Mg²⁺ or Mn²⁺. At higher concentrations, both ions inhibited epinephrine-stimulated, but not basal or fluoride-stimulated activities. Approximately equivalent hormonal stimulation was obtained with maximal stimulating concentrations of epinephrine, isoproterenol, glucagon, and prostaglandin E₁. Norepinephrine was less stimulatory. Only catecholamine-stimulated activities were inhibited by propranolol (10^?5M). The data suggest that catecholamines stimulate frog liver adenylate cyclase through interactions with β adrenergic receptors. The adenylate cyclase in frog liver differs from its mammalian counterpart in its response to temperature and maximally stimulatory concentrations of hormones.     

Effects of prostaglandin H2, prostaglandin E2, and arachidonic acid on parathyroid hormone and antidiuretic hormone activation of rat kidney adenylate cyclase

These experiments examine interactions of arachidonic acid; the substrate for prostaglandin cyclooxygenase, prostaglandin (PG)H₂, a key endoperoxide intermediate in prostaglandin synthesis; and prostaglandin (PG)E₂, an important prostaglandin produced within the kidney; with adenylate cyclase activity in renal cortex, outer medulla, and inner medulla. In addition, the effects of arachidonic acid, PGH₂, and PGE₂ on parathyroid hormone (PTH) activation of adenylate cyclase in cortex, and of antidiuretic hormone (ADH) activation of that enzyme in outer and inner medulla are examined. Arachidonic acid elicited a concentration-dependent inhibition of basal and PTH-stimulated adenylate cyclase activity in renal cortex. Concentration-dependent inhibition by arachidonic acid of basal and ADH-stimulated adenylate cyclase activity was observed in outer and inner medulla. PGH₂ inhibited basal activity in all three areas of the kidney. There was also inhibition by PGH₂ of medullary ADH and cortical PTH stimulation. PGE₂ stimulated adenylate cyclase in all three areas. PGE₂ had no effect upon PTH stimulation in cortex and was additive with ADH in outer and inner medulla. PGE₂ stimulation was inhibited by arachidonic acid, and this inhibition seemed competitive. Inhibition by both arachidonic acid and PGH₂ was not destructive. Experiments with [1-¹⁴C]arachidonic acid and indomethacin suggest that the inhibition by arachidonic acid was actually mediated by arachidonic acid and not a metabolite. Both PGH₂ and arachidonic acid inhibition was independent of phosphodiesterase. This activation by product, PGE₂, and inhibition by its precursors, arachidonic acid and PGH₂, provide a possible mechanism by which the prostaglandin system could modulate adenylate cyclase responsiveness to hormonal activation.     

Prostaglandins of the one,two, and three series affect blood pressure in the American bullfrog, Rana catesbeiana

The effects of prostagandins (PGs), and three potential prostaglandin precursors, were studied on blood pressure and heart rate of the American bullfrog, Rana catesbeiana. Bullfrogs were chronically cannulated with a T cannula in the right sciatic artery. The mean systemic arterial blood pressure (SAP) prior to infusion was 20.4 ± 1.1 mm Hg. Mean preinfusion systolic and diastolic pressures were 23.9 ± 1.4 and 17.1 ± 0.9 mm Hg, respectively. Mean preinfusion heart rate was 41.1 ± 0.5 beats/min. Of the PGs tested, PGI₂ was a potent hypotensive agent, with effects at 0.03 μg/kg bw. PGE₂ was more potent than PGE₃, and PGE₁. PGA₁ and PGA₂ were the least potent, and were ineffective at doses below 100 μg/kg bw. PGF_2α was the most potent hypertensive agent tested, with thromboxane B₂ less potent. All compounds tested elevated heart rate, with PGE₂ the most effective. The prostaglandin precursors, eicosatrienoic acid, arachidonic acid, and eicosapentaenoic acid (2000 μg/kg bw) all decreased blood pressure by approximately 25%. The decrease was attenuated by indomethacin (4 mg/kg bw). These results indicate that the bullfrog utilizes all three hypotensive activity. The ability of the bullfrog to utilize several substrates makes it a good choice for comparative studies on prostaglandin synthesis.     

C3 inhibition with pegcetacoplan in subjects with paroxysmal nocturnal hemoglobinuria treated with eculizumab

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired, life-threatening hematologic disease characterized by chronic complement-mediated hemolysis and thrombosis. Despite treatment with eculizumab, a C5 inhibitor, 72% of individuals remain anemic. Pegcetacoplan (APL-2), a PEGylated C3 inhibitor, has the potential to provide more complete hemolysis control in patients with PNH. This open-label, phase Ib study was designed to assess the safety, tolerability, and pharmacokinetics of pegcetacoplan in subjects with PNH who remained anemic during treatment with eculizumab. Pharmacodynamic endpoints were also assessed as an exploratory objective of this study. Data are presented for six subjects in cohort 4 who received treatment for up to 2 years. In total, 427 treatment-emergent adverse events (TEAEs) were reported, 68 of which were possibly related to the study drug. Eight serious TEAEs occurred in two subjects; three of these events were considered possibly related to the study drug. Pegcetacoplan pharmacokinetic concentrations accumulated with repeated dosing, and steady state was reached at approximately 6-8 weeks. Lactate dehydrogenase levels were well controlled by eculizumab at baseline. Pegcetacoplan increased hemoglobin levels and decreased both reticulocyte count and total bilirubin in all six subjects. Improvements were observed in Functional Assessment of Chronic Illness Therapy Fatigue scores. Two subjects discontinued for reasons unrelated to pegcetacoplan. All four subjects who completed the study transitioned to pegcetacoplan monotherapy following eculizumab discontinuation and avoided transfusions. In this small study, pegcetacoplan therapy was generally well-tolerated, and resulted in an improved hematological response by achieving broad hemolysis control, enabling eculizumab discontinuation.     

Catecholamine effects on blood pressure and heart rate in the American bullfrog, Rana catesbeiana

The effects of catecholamines and adrenergic blocking agents were studied in vivo on the blood pressure and heart rate of the unanaesthetized American bullfrog, Rana catesbeiana. Bullfrogs were chronically cannulated with a T cannula in the right sciatic artery. The mean systemic arterial blood pressure prior to the infusion of catecholamines was 18.5 ± 1.5 mm Hg. Mean preinfusion heart rate was 30.9 ± 2.0 beats/min. Epinephrine elicited the largest increase in blood pressure, with an accompanying decrease in heart rate. Norepinephrine and phenylephrine were less effective. Isoproterenol was the only catecholamine tested which elevated heart rate in a dose-dependent manner. It had no effect on blood pressure. The beta adrenergic antagonist, propranolol, blocked the increase in heart rate elicited by isoproterenol but had no effect on the blood pressure increases elecited by the other catecholamines. The alpha adrenergic antagonist, phentolamine, partially blocked the blood pressure increase by epinephrine, norepinephrine, and phenylephrine as well as the elevation of heart rate by isoproterenol. Atropine alone elevated heart rate 19 ± 3 beats/min, and prevented slowing of the heart due to epinephrine, norepinephrine, and phenylephrine. Stimulatory effects of epinephrine on heart rate were observed only after atropine had been administered. Beta adrenergic receptors, therefore, appear to function in heart rate regulation; however, the predominant effect of catecholamines is reflex slowing of the heart due to stimulation of the vagus nerve. In contrast, the alpha receptor, stimulated by epinephrine, appears to be the main adrenergic receptor controlling blood pressure changes.     

Neuregulin-1 attenuates neointimal formation following vascular injury and inhibits the proliferation of vascular smooth muscle cells

Neuregulin-1 (NRG-1) is expressed in vascular endothelial cells, and its receptors are localized to the underlying smooth muscle cells. However, the role of NRG-1 in vascular function and injury is largely unknown. First, the expression of NRG-1 and its receptors (erbB receptors) was analyzed after balloon injury to the rat carotid artery. NRG-1 and erbB expression levels were low in uninjured vessels; however, NRG-1 and erbB4 were upregulated following injury. We then examined the effect of NRG-1 on neointimal formation following balloon injury. NRG-1 was administered by tail-vein injection prior to injury and every 2 days following injury. Two weeks after injury, NRG-1-treated animals demonstrated a 50% reduction in lesion size compared with controls receiving the vehicle. To examine possible mechanisms for NRG-1 action, we examined its effects on vascular smooth muscle cell (VSMC) function. Rat VSMC cultures were pretreated with NRG-1 for 24 h and then stimulated with platelet-derived growth factor. NRG-1 significantly decreased platelet-derived growth factor-stimulated VSMC proliferation and migration. These findings suggest that NRG-1 may be a novel therapeutic candidate for the treatment of restenosis and atherosclerosis.     

Complement in immune thrombocytopenia (ITP): The role of complement in refractory ITP

Immune thrombocytopenia (ITP) is a disorder characterized by low platelets due to increased clearance and decreased platelet production. While ITP has been characterized as an acquired disorder of the adaptive immune system, the resulting platelet autoantibodies provide ancillary links to the innate immune system via antibody interaction with the complement system. Most autoantibodies in patients with ITP are of the IgG1 subclass, which can be potent activators of the classical complement pathway. Antibody-coated platelets can initiate complement activation via the classical pathway leading to both direct platelet destruction and enhanced clearance of C3b-coated platelets by complement receptors. Similar autoantibody interactions with bone marrow megakaryocytes can also result in complement injury and ineffective thrombopoiesis. The development of novel therapeutic complement inhibitors has revived interest in the role of complement in autoantibody-mediated disorders, such as ITP. A recent early-phase clinical trial of a classical complement pathway inhibitor has demonstrated efficacy in a subset of ITP patients refractory to conventional immune modulation. In this review, we will analyse the role of complement in refractory ITP.