Phospholipid metabolism, calcium flux, and the receptor-mediated induction of chemotaxis in rabbit neutrophils.

Rabbit neutrophils were stimulated with the chemotactic peptide fMet-Leu-Phe in the presence of the methyltransferase inhibitors homocysteine (HCYS) and 3-deazaadenosine (3-DZA). HCYS and 3-DZA inhibited chemotaxis, phospholipid methylation, and protein carboxymethylation in a dose-dependent manner. The chemotactic peptide-stimulated release of [14C]arachidonic acid previously incorporated into phospholipid was also partially blocked by the methyltransferase inhibitors. Stimulation by fMet-Leu-Phe or the calcium ionophore A23187 caused release of arachidonic acid but not of previously incorporated [14C]-labeled linoleic, oleic, or stearic acids. Unlike the arachidonic acid release caused by fMet-Leu-Phe, release stimulated by the ionophore could not be inhibited by HCYS and 3-DZA, suggesting that the release was caused by a different mechanism or by stimulating a step after methylation in the pathway from receptor activation to arachidonic acid release. Extracellular calcium was required for arachidonic acid release, and methyltransferase inhibitors were found to partially inhibit chemotactic peptide-stimulated calcium influx. These results suggest that methylation pathways may be associated with the chemotactic peptide receptor stimulation of calcium influx and activation of a phospholipase A2 specific for cleaving arachidonic acid from phospholipids.

Responses of heart ornithine decarboxylase and adrenal catecholamines to methadone and sympathetic stimulants in developing and adults rats.

Sympathetic stimulation elevates heart ornithine decarboxylase (ODC) activity in adult rats, an effect which is followed by cardiac hypertrophy. In developing animals, adrenergic agonists play a role in cardiac growth and differentiation. Methadone administration to adult rats stimulated heart ODC, and this stimulation could be blocked by the sympatholytic agents chlorisondamine, reserpine or propranolol, suggesting that the effect is mediated via central stimulation of sympathetic nerves. With repeated methadone administration, tolerance developed to the heart ODC stimulation by methadone, eventually resulting in decreased heart ODC accompanied by deficits in heart weight. The sensitivity of the mature heart and adrenal medulla to the sympathetic stimulants insulin, nicotine or isoproterenol was virtually unchanged during long-term methadone administration, indicating that tolerance of the heart ODC response to methadone did not result from subsensitivity of the efferent sympathetic pathway or the heart ODC response system. In normally developing rats, responses to insulin-induced reflex stimulation of sympathetic nerve supplies to the heart and adrenal medulla did not occur until 8 days of postnatal age; however, daily treatment of pups with methadone begun the day after birth accelerated the development of functiona heart and adrenal medullary responses to insulin such that responses were obtained by 4 days of age. The cardiac responses to nicotine developed somewhat differently, with significant ODC stimulation first appearing at 12 days in controls and at 8 days in methadone-treated pups. In the adrenal gland, a tissue in which responses to nicotine do not depend on an intact nerve supply a catecholamine secretory response to nicotine was observed at 4 days of age in controls. These data suggest that methadone treatment accelerates the development of functional sympathetic innervation of heart and adrenal medulla, with resultant abnormalities in cardiac muscle growth and differentiation.

Maturation of sympathetic neurotransmission in the rat heart. IV. Effects guanethidine-induced sympathectomy on neonatal development of synaptic vesicles, synaptic terminal function and heart growth.

Sympathetic nerve input has been proposed to regulate cardiac growth and differentiation. In the present study, this hypothesis was tested by giving the neurotoxic adrenergic neuron blocking agent, guanethidine (50 mg/kg, s.c.), daily to rats for 21 consecutive days to produce long-term peripheral sympathectomy in neonatal rats. Ontogeny of the sympathetic nerve terminal was measured by the ablity of synaptic vesicle preparations to take up radiolabeled norepinephrine, and heart growth in the sympathectomized animals was monitored by organ weight as well as by RNA and protein synthesis. Guanethidine treatment produced a massive sympathectomy, as synaptic vesicle development was totally arrested; the functional consequence of this treatment was confirmed by the attenuation of chronotropic responses to tyramine, a drug which acts by displacement of norepinephrine from the noradrenergic terminal. Despite the clear-cut effectiveness of guanethidine to prevent formation of functional sympathetic innervation of the heart, no significant alterations in heart growth or RNA and protein synthetic capabilities were observed in the developing rats. These results suggest that the presence of sympathetic innervaton is not obligatory for normal growth of the heart to occur.

Bradykinin stimulates phospholipid methylation, calcium influx, prostaglandin formation, and cAMP accumulation in human fibroblasts.

The biochemical events that lead to bradykinin stimulation of cAMP accumulation in human fibroblasts were examined. Treatment of human fibroblasts with bradykinin increases phospholipid methylation, Ca2+ influx, arachidonic acid release, prostaglandin formation, and cAMP content. The dose-response curves of bradykinin for the increase in the above changes were similar. In human fibroblasts, exogenous arachidonic acid was mainly incorporated into phosphatidylcholine, followed by phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol. Bradykinin caused a release of arachidonic acid from methylated phospholipids (phosphatidylcholine) and phosphatidylinositol. 3-Deazaadenosine, a methyltransferase inhibitor, almost completely inhibited bradykinin-stimulated phospholipid methylation and Ca2+ influx and partially reduced arachidonic acid release and prostaglandin formation but had no effect on cAMP formation. Mepacrine, a phospholipase inhibitor, blocked bradykinin-induced arachidonic acid release, prostaglandin release, and cAMP accumulation. Indomethacin, a cyclooxygenase inhibitor, blocked the effect of bradykinin on cAMP accumulation. Prostaglandins E1 and E2, but not F2 alpha, increased accumulation of cAMP. These observations indicate that bradykinin generates cAMP via arachidonic acid release and subsequent formation of prostaglandins. Our findings suggest that arachidonic acid can arise from either phosphatidylcholine synthesized by the methylation pathway or phosphatidylinositol.