Renovascular actions of angiotensin II in the isolated kidney of the rat: relationship to lipoxygenases.

Several actions of angiotensin II have been linked to metabolism of arachidonic acid by lipoxygenases. To evaluate the importance of this interaction intrarenally we tested the effect of three different lipoxygenase inhibitors, BW755c (50 microM), a dual lipoxygenase-cyclooxygenase inhibitor, MK447 (200 microM), a nonselective lipoxygenase inhibitor which can stimulate cyclooxygenase, and baicalein (1 microM), a highly selective 12-lipoxygenase inhibitor, on angiotensin II-evoked hemodynamic changes in the rat isolated kidney, perfused with oncotic agents. Kidneys were pretreated with indomethacin (10 microM) to exclude participation of cyclooxygenase-dependent arachidonate products. Renal perfusion pressure was kept constant at 90 mm Hg by continuous adjustments in perfusate flow rate. Inhibition of cyclooxygenase alone produced a transient potentiation of the vasoconstrictor response to angiotensin II without altering GFR. On the other hand, the lipoxygenase inhibitors attenuated the angiotensin II-induced increase in renal vascular resistance by approximately 50% and promoted an increase in GFR above that of kidneys infused with angiotensin II in the presence of only indomethacin. Base-line values were essentially unchanged by lipoxygenase inhibition. Furthermore, the vasoconstrictor response to the thromboxane/endoperoxide agonist U46619 was unaffected. We conclude that products of the lipoxygenase pathway, arising within the kidney, contribute to the renal hemodynamic effects of angiotensin II.

Cells in culture from rabbit medullary thick ascending limb of Henle's loop.

Freshly isolated cells obtained from the medullary segment of the rabbit thick ascending limb of Henle's loop (mTALH) metabolize arachidonic acid (AA) primarily by the cytochrome P-450 monooxygenase pathway forming several products; a vasorelaxant and an inhibitor of Na+-K+-ATPase have been identified. These studies have been extended to mTALH cells in culture. The ability of cells isolated from 1-mo-old rabbits to grow in culture far surpassed that of cells isolated from adult rabbits, whereas similar cytochrome P-450-dependent AA metabolites were produced by freshly isolated cells from rabbits of both ages. Three-week-old mTALH cultures formed ouabain-sensitive "domes" when grown on plastic surfaces and developed transepithelial voltages (4.7 + 1.2 mV, n = 6) when grown on gas-permeable surfaces. Electron microscopy of the cells showed typical mTALH cell characteristics. The presence of Tamm-Horsfall protein, a surface membrane protein of mTALH cells, in 90-95% of the cells confirmed the homogeneity of the cultures. Although several environmental manipulations were tested, mTALH cells in culture did not produce the same cytochrome P-450-dependent AA metabolites as those produced by mTALH cells before culture. However, a cytochrome P-450-dependent AA metabolite that differs from the AA metabolites formed by freshly isolated mTALH cells was produced by hemin-treated mTALH and heterogenous cell cultures.

Trophic influence of sympathetic neurons on the cardiac alpha-adrenergic response requires close nerve-muscle association.

In tissue culture, sympathetic neurons exert a trophic effect on myocardial cells to alter the alpha-adrenergic response from positive to negative chronotropism. We hypothesized that this neurotrophic effect might be a result of the release of a humoral substance into the bulk phase of the culture medium by the neurons. The negative chronotropic response to alpha stimulation persisted in nerve-muscle cultures pretreated with the muscarinic antagonist atropine, indicating that acetylcholine is not the trophic agent. Further, variations in the concentration of norepinephrine, epinephrine or dopamine in the nerve-muscle culture media do not account for the presence of a negative chronotropic response to alpha stimulation. Finally, muscle cells grown in the same petri dish with innervated muscle cells, to allow conditioning of the muscle cell environment by the neurons, do not acquire a negative chronotropic response to alpha stimulation. The results of this study clearly demonstrate that this trophic effect is not due to the release of a humoral substance into the bulk phase of the culture medium, but requires close nerve-muscle association. This suggests that the trophic effect may involve localized release and transsynaptic transfer of a chemical substance other than a neurotransmitter.

Acquisition by innervated cardiac myocytes of a pertussis toxin-specific regulatory protein linked to the alpha 1-receptor.

During development, the chronotropic response of rat ventricular myocardium to alpha 1-adrenergic stimulation changes from positive to negative. The alpha 1-agonist phenylephrine increases the rate of contraction of neonatal rat myocytes cultured alone but decreases the rate of contraction when the myocytes are cultured with functional sympathetic neurons. The developmental induction of the inhibitory myocardial response to alpha 1-adrenergic stimulation in intact ventricle and in cultured myocytes was shown to coincide with the functional acquisition of a substrate for pertussis toxin. A 41-kilodalton protein from myocytes cultured with sympathetic neurons and from adult rat myocardium showed, respectively, 2.2- and 16-fold increases in pertussis toxin-associated ADP-ribosylation (ADP, adenosine diphosphate) as compared to controls. In nerve-muscle cultures, inhibition of the actions of this protein by pertussis toxin-specific ADP-ribosylation reversed the mature inhibitory alpha 1-adrenergic response to an immature stimulatory pattern. The results suggest that innervation is associated with the appearance of a functional pertussis toxin substrate by which the alpha 1-adrenergic response becomes linked to a decrease in automaticity.

Neuronal regulation of the development of the alpha-adrenergic chronotropic response in the rat heart.

During development, there are changes in the response of automatic cardiac fibers to alpha-adrenergic agonists. In neonatal rat ventricle, in vitro phenylephrine (1 X 10(-8) M) induces an increase in automatic rate from 115 +/- 12 (mean +/- SEM) to 168 +/- 10 beats/min, P less than 0.05. In contrast, in adult rat ventricle, the rate decreases from 36 +/- 8 to 12 +/- 12 beats/min, P less than 0.05. At both ages, the response is attenuated by the alpha 1-antagonist, prazosin (1 X 10(-6) M). We used cultures of neonatal rat myocytes to determine whether maturation of innervation contributes to the ontogeny of this response. All non-innervated cultures showed a positive chronotropic response to alpha-stimulation; phenylephrine (1 X 10(-8) M) increased the rate from 40 +/- 2 to 52 +/- 2 beats/min, P less than 0.05. In contrast, 60% of the myocytes innervated with sympathetic neurons showed a decrease in rate in response to phenylephrine, from 78 +/- 6 to 67 +/- 6 beats/min, P less than 0.05. The negative chronotropic effect of phenylephrine did not result from the release of acetylcholine or adenosine, or the inhibition of presynaptic norepinephrine release by phenylephrine. Furthermore, exposure to neuronal norepinephrine is not responsible for the alteration in muscle cell responsiveness. In conclusion, we have demonstrated the modulation of the myocardial response to alpha-adrenergic stimulation by the occurrence of innervation in tissue culture. This provides an explanation for the previously identified ontogenetic change in alpha-adrenergic effects on intact cardiac fibers from excitation to inhibition.

Effects of synthetic parathyroid hormone on hemodynamics and regional blood flows.

In pentobarbital anesthetized dogs, synthetic bovine parathyroid hormone, containing the amino terminal 34 amino acids (bPTH-(1-34] in doses of 0.1-0.8 microgram/kg i.v. caused dose-related decrease in arterial blood pressure, increase in cardiac output and prominent reduction of total peripheral resistance. Marked increase of coeliac, coronary and renal blood flows and decrease of vascular resistances in these beds occurred. Mesenteric and iliac blood flows usually decreased. Changes in mesenteric resistance were minimal while iliac resistance increased substantially. The increase of renal blood flow was still obvious 10-20 min or longer after arterial blood pressure had returned to control levels. Mesenteric and iliac vasoconstriction were attributable to reflex increase in sympathetic activity since some decrease in resistances in these beds were seen in response to bPTH-(1-34) after ganglionic blockade. Renal vasodilation was not related to a prostaglandin mechanism as similar dilation occurred after prostaglandin synthesis inhibition with indomethacin. In summary, bPTH-(1-34) has prominant effects on the circulation, and does not affect all vascular beds similarly. The exact mechanism of the vasodilating action of bPTH remains to be elucidated.