Health care tips for your professional corporation. II. Or where there is a buy-sell agreement and an employment agreement, there is a way.

In my earlier article, I discussed the importance of the Buy-Sell Agreement and the Employment Contract in the professional corporation (PC) environment. In that article the discussion was centered primarily upon the Buy-Sell Agreement and its structure. In this article, my comments will focus upon the shareholder Employment Contract with suggested provisions for language to enhance its efficacy.

Peripheral opioidergic mechanisms do not mediate naloxone's pressor effect in the conscious rabbit.

We tested the hypothesis that the pressor effect of naloxone during acute hemorrhagic hypotension is mediated in part at peripheral sites. Experiments were performed in conscious, chronically prepared rabbits. First, we compared the hemodynamic response to peripheral injections of naloxone and naloxone methobromide during acute hemorrhagic hypotension. Naloxone methobromide, which does not enter the central nervous system, produced a lesser pressor effect than naloxone. Second, we looked for peripheral effects of naloxone after close-arterial injection into the hindquarter vasculature. Unlike i.v. injections, close-arterial injection of naloxone did not produce any significant hemodynamic changes during hemorrhagic hypotension. Finally, we compared the capacities of naloxone and naloxone methobromide to block the peripherally mediated cardiovascular response to i.v. methionine-enkephalin in nonhemorrhaged animals. The potency of the two compounds, in terms of their blockade of this peripherally mediated response, was similar. The results of the present study do not support a predominant peripheral role for naloxone during acute hemorrhagic hypotension in conscious rabbits.

Analysis of serotonergic mechanisms underlying benzamide-induced gastroprokinesis.

Serotonin (5-HT) receptors in the myenteric plexus mediate contractility in vitro and may regulate gastric emptying in vivo. This report examines the pharmacology of three benzamides, ML-1035 (4-amino-5-chloro-2-[2-(methylsulfinyl)-ethoxy]-N-[2- (diethylamino)ethyl]-benzamide hydrochloride), metoclopramide and cisapride, in studies which address the serotonergic mechanisms underlying benzamide-induced gastroprokinesis. All three compounds had high affinity at the 5-HT3 receptor as they displaced the 5-HT3 antagonist [3H]GR65630 from cortical membranes (Ki = 156, 232 and 1711 nM for ML-1035, metoclopramide and cisapride, respectively) and blocked the 5-HT-induced Bezold-Jarisch reflex, although cisapride was much less active in this experiment. Receptor selectivity was also compared at 5-HT1, 5-HT2, and dopamine D2 receptors in which no displacement was observed that was common to all agents. All benzamides elicited a 5-HT4-like agonist response as they enhanced field-stimulated neurogenic contractions in ileum (EC50 = 1.4, 1.6 and 0.013 microM for ML-1035, metoclopramide and cisapride, respectively). ICS 205-930, a proposed 5-HT4 antagonist, competitively antagonized this response for ML-1035 (Kb = 1.6 microM) whereas atropine blocked the twitch response and any additional responses to ML-1035. In vivo, ML-1035 and metoclopramide increased gastric emptying (IC50 = 0.87 and 3.09 mg/kg i.p., respectively). Thus, the benzamides activate a 5-HT4 receptor in the ileum which increases cholinergic contractions and may be one mechanism by which these agents increase gastric emptying.

Renin-angiotensin system and opioids during acute hemorrhage in conscious rabbits.

We measured changes in plasma renin activity (PRA) and used angiotensin-converting enzyme blockade with captopril to evaluate the role of the renin-angiotensin system during hemorrhage and after opioid receptor blockade in conscious rabbits. The increase in PRA after nonhypotensive hemorrhage was not statistically significant. PRA increased sixfold after a hypotensive hemorrhage to a mean arterial pressure less than 40 mmHg. This increase was statistically significant. Captopril altered the hemodynamic response to hemorrhage. The normal increase in vascular resistance early in hemorrhage was reduced by captopril pretreatment. After a critical blood loss, arterial pressure and heart rate decreased in both groups. The blood loss required to decrease mean arterial pressure to less than 40 mmHg was approximately 25% less in animals pretreated with captopril. The characteristic decrease in vascular resistance coincident with the onset of hypotension was still present after captopril pretreatment. Injection of naloxone or saline during acute hemorrhagic hypotension did not affect PRA. However, recovery of blood pressure after naloxone or saline was attenuated by converting-enzyme blockade. This attenuation was due primarily to a reduction in spontaneous recovery (i.e., recovery after the control saline injection) and not to a reduction in the response to naloxone. We tested whether this effect of captopril might be due to an interaction of ANG II and catecholamines. The plasma norepinephrine (NE) response to naloxone was statistically similar with and without captopril. In contrast, the response to exogenous NE after hypotensive hemorrhage was significantly reduced by captopril pretreatment. Captopril apparently did not alter baroreflex sensitivity but did reset the baroreflex to lower pressure levels during naloxone's pressor response.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological and thrombolytic properties of fibrolase--a new fibrinolytic protease from snake venom.

Fibrolase, a direct-acting fibrinolytic enzyme has been shown to cleave primarily the A alpha and B beta chains of human fibrin. We have previously reported that fibrolase also exhibits fibrinogenolytic activity and acts mainly as an alpha-chain fibrinogenase. In contrast to the action of streptokinase (plasminogen activator), fibrolase does not activate plasminogen. In vitro thrombolytic efficacy of fibrolase was determined by monitoring the release of radiolabel from iodinated fibrin and human blood clots. Fibrolase effectively digested the clots in a dose-dependent manner. The in vivo efficacy of fibrolase was evaluated in an animal model of arterial thrombosis. Fibrolase was found to be efficacious at dissolving femoral arterial clots following a single intravenous bolus administration. Time to reperfusion was dose dependent and similar to that observed with streptokinase. No adverse effects on blood pressure and heart rate were observed.

Role of adrenal medulla in hemodynamic response to hemorrhage and naloxone.

We tested the hypothesis that enkephalins or some other compound(s) released by the adrenal medulla during hemorrhage were responsible for the resultant hypotension. We compared the hemodynamic and plasma catecholamine responses to hemorrhage and subsequent opioid receptor blockade with naloxone in intact, adrenal-denervated (ADD), and adrenalectomized (ADX) rabbits. The studies were done in conscious, chronically prepared, male New Zealand White rabbits. The hemodynamic response to hemorrhage was not different among the three groups. Plasma norepinephrine (NE) increased early in hemorrhage in all groups. In the ADD and ADX animals, NE decreased significantly at the transition to hypotension, suggesting decreased release of NE by peripheral sympathetic nerves as a possible cause of the decrease in pressure. In the intact group, NE did not decrease but reached a plateau possibly due to the release of some NE by the adrenal medulla, which obscured the decreased release by sympathetic nerves. The pressor response to naloxone, though present in all groups, was attenuated by adrenalectomy or adrenal denervation. The plasma NE response to naloxone was similar in all groups and involved a two- to threefold increase after naloxone. We conclude that enkephalins or any other compounds released by the adrenal gland are not responsible for the acute hemodynamic changes during hemorrhage in the conscious rabbit. However, some substance(s) released by the adrenal medulla, perhaps epinephrine, does play a role in naloxone's pressor effect, since this is reduced by adrenalectomy or adrenal denervation.

Acceleration of coronary collateral development by heparin in conscious dogs.

We evaluated whether heparin pretreatment accelerates the development of coronary collateral vessels induced by repeated, brief coronary occlusions. Sixteen dogs were instrumented for the measurement of subendocardial segment length in the area perfused by the left circumflex coronary artery (LCCA), LCCA flow and left ventricular pressure. An externally inflatable pneumatic occluder was placed around the LCCA. Two min coronary occlusions (CO) at rest were repeated hourly until there was no reduction in ischemic segment systolic shortening at the end of CO and negligible reactive hyperemia following the release of CO. Eight control dogs developed collaterals sufficient for resting myocardial oxygen requirements in the LCCA region by 129 +/- 45 (SD) CO. The remaining 8 dogs given heparin daily developed collaterals by 81 +/- 33 CO (p less than 0.05). Thus, in the presence of severe myocardial ischemia known to promote collateralization, heparin accelerated the development of coronary collaterals.

Decreased vascular resistance after intra-arterial injection of [met]enkephalin in the hindquarters of conscious rabbits.

The hemodynamic effects of i.a. [met]enkephalin were studied in the hindquarter vasculature of chronically prepared, conscious rabbits. A new method allowed i.a. injection while simultaneously measuring blood pressure and blood flow to this vascular bed. [Met]enkephalin produced a dose-dependent (3-300 micrograms/kg) decrease in hindquarter vascular resistance (18-42% change from base line). The duration of the response ranged from 28 sec to over 2 min. Heart rate decreased 16 to 45% over the same dose range but returned to preinjection levels in 5 sec. Only the bradycardia was abolished by pretreatment with atropine methyl nitrate. All hemodynamic changes were eliminated or significantly reduced after pretreatment with the ganglionic blocking agent, chlorisondamine hydrochloride. The opioid antagonist, naloxone hydrochloride, abolished the hemodynamic effects of [met]enkephalin. Resistance decreases in the mesenteric vasculature were coincident with those in the hindquarters. The time to onset of the response was delayed when [met]enkephalin was injected i.v. These data indicate activation of a reflex originating in the hindquarters that resulted in opioid-dependent increased efferent parasympathetic activity to the heart and decreased sympathetic tone to at least two vascular beds.

Cardiovascular responses to hemorrhage and naloxone in conscious barodenervated rabbits.

The hemodynamic and plasma catecholamine response to hypotensive hemorrhage and subsequent opioid receptor blockade with naloxone were evaluated before and after complete sinoaortic denervation (SAD). This study was done to test the general hypothesis that opioid-mediated failure of the baroreflex accounts for the hypotension of hemorrhage. The specific hypothesis we tested was that SAD would abolish the pressor effect of opioid receptor blockade with naloxone. The studies were done in conscious chronically prepared rabbits. Hemorrhage of 12 ml/kg did not change mean arterial blood pressure in intact animals due to a compensatory increase in heart rate and vascular resistance. When blood loss exceeded 12 ml/kg, pressure decreased abruptly due to a decrease in vascular resistance. Plasma norepinephrine (NE) and epinephrine (E) were higher after hemorrhage than before. Plasma E levels increased almost 70 times. After SAD, mean blood pressure began to decrease at the beginning of hemorrhage, the heart rate increase was abolished, and vascular resistance decreased throughout the blood loss. Plasma NE was no different after hemorrhage than before. Plasma E increased, but the increase was only fivefold. Naloxone increased mean arterial blood pressure, vascular resistance, cardiac index, and plasma NE before and after SAD. The increases in blood pressure and plasma norepinephrine were significantly greater after SAD. Therefore the pressor effect of naloxone in this model is not due to increased baroreflex sensitivity. Rather, intact baroreflexes buffer naloxone's effects.

Effects of ouabain on the nerve stimulation-evoked release of norepinephrine from the isolated perfused guinea-pig heart.

The isolated perfused guinea-pig heart prelabeled with (-)-[7-3H]-norepinephrine was used to examine the effects of increasing concentrations of ouabain on the sympathetic nerve stimulation-evoked release of endogenous norepinephrine and [3H]norepinephrine in the presence and absence of physostigmine or atropine. The overflow of norepinephrine and [3H]norepinephrine from guinea-pig hearts was measured during postganglionic stimulation of the cardiac accelerator fibers (5 Hz for 60 sec, 2 msec duration, for 300 pulses). Perfusion with 10(-7) M ouabain for 20 min had no effect on the release of norepinephrine or [3H]norepinephrine after nerve stimulation. However, perfusion with either 10(-6) or 3 X 10(-6) M ouabain for 15 min resulted in a significant decrease in the nerve stimulation-evoked release of norepinephrine (44.6 +/- 2.24 and 44.0 +/- 2.17%, respectively) and [3H]norepinephrine (43.8 +/- 1.62 and 44.9 +/- 2.16%, respectively) compared with previous control outputs. Perfusion of hearts with physostigmine (10(-6) M), an acetylcholinesterase inhibitor, or atropine (3 X 10(-6) M), a muscarinic blocking agent, did not alter the release of norepinephrine or [3H]norepinephrine after nerve stimulation. Perfusion with physostigmine during perfusion with 10(-6) M ouabain resulted in a decrease in the release of norepinephrine and [3H]norepinephrine only slightly greater than 10(-6) M ouabain alone, which was not significant, but the release of norepinephrine during stimulations performed after a 45-min washout of 10(-6) M ouabain was decreased significantly when 10(-6) M physostigmine was present. Perfusion of hearts with atropine during perfusion with either 10(-6) or 3 X 10(-6) M ouabain reversed the inhibitory effect of ouabain on the release of norepinephrine and [3H]norepinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous opiate peptides may limit norepinephrine release during hemorrhage.

The involvement of the sympathetic nervous system in the cardiovascular response to hemorrhage and subsequent opiate receptor blockade was studied in conscious rabbits. Plasma catecholamines were measured by high-pressure liquid chromatography to indirectly assess sympathetic activity. Arterial blood samples were drawn at four times during the experiment: 1) before hemorrhage; 2) after a 15% blood loss; 3) after mean arterial blood pressure decreased to less than 40 mm Hg; and 4) 2 min after an i.v. injection of naloxone (3 mg/kg) or saline. Rapid removal of 15% of the total blood volume (approximately equal to 8 ml/kg) increased heart rate and plasma norepinephrine. Plasma epinephrine and blood pressure remained at control levels. Further hemorrhage (approximately equal to 16 ml/kg) produced a sudden decrease in blood pressure and a large increase in plasma epinephrine. Plasma norepinephrine was not significantly different from the previous sample. Subsequent injection of naloxone significantly increased plasma norepinephrine and blood pressure compared to the saline-treated group. Plasma epinephrine was similar in the two groups. These studies suggest that naloxone may exert its pressor effect during hemorrhagic hypotension in the conscious rabbit by blocking a naturally occurring, opiate peptide-mediated inhibition of norepinephrine release. The results are consistent with a peptidergic limit on sympathetic activity being responsible for the decrease in blood pressure seen during acute hemorrhage.

Effects of ethylketocyclazocine on adrenergic transmission in the isolated perfused cat spleen.

The pre- and postsynaptic effects of ethylketocyclazocine (EKC), a putative kappa opiate receptor agonist, on sympathetic adrenergic neurotransmission were investigated using the isolated perfused cat spleen. Perfusion of spleens with EKC (10(-7) and 10(-6)M) produced a modest dose-dependent inhibition of nerve-stimulation-mediated overflow of norepinephrine (NE) and total 3H. In contrast, EKC (10(-4)M) produced an increase in the release of NE, and total 3H overflow. Perfusion with EKC (10(-5) and 10(-4)M) resulted in a dose-dependent inhibition of the postsynaptic response to stimulation-mediated release of NE. Likewise, EKC inhibited splenic contraction due to exogenously administered NE which was not antagonized by naloxone. These results suggest that EKC exerts a direct effect on peripheral adrenergic neurotransmission not mediated via classical kappa opiate receptors.

Modulation of peripheral adrenergic neurotransmission by methionine-enkephalin.

The isolated perfusion cat spleen prelabeled with [3H]norepinephrine was used to study the effects of morphine and Met-enkephalin on the exocytotic release of norepinephrine from sympathetic adrenergic neurons after nerve stimulation. The overflow of endogenous norepinephrine, total 3H and dopamine-beta-hydroxylase (DBH) from cat spleens was measured during postganglionic stimulation of the splenic nerve. Perfusion of spleens with Met-enkephalin (10(-8)-10(-5) M) produced a dose-dependent decrease in the release of endogenous norepinephrine upon nerve stimulation. These changes were paralleled by significant dose-dependent Met-enkephalin-induced decreases in the nerve stimulation-mediated release of total 3H, DBH and in the perfusion pressure. However, perfusion of spleens with morphine (10(-7)-10(-4) M) produced no significant changes in the release of endogenous norepinephrine, total 3H or DBH after nerve stimulation at 5 Hz. Morphine (10(-7)-10(-4) M) also had no significant effects on the contraction of the splenic capsule. Perfusion of spleens with naloxone (10(-6) M), a pure narcotic antagonist, did not alter the release of endogenous norepinephrine, total 3H, DBH or perfusion pressure. However, perfusion with naloxone (10(-6) M) plus Met-enkephalin (10(-6)-10(-5) M) antagonized the inhibitory effects of Met-enkephalin. These findings support the hypothesis that the opiate receptor population in peripheral tissues are heterogenous and that Met-enkephalin depresses exocytotic release of norepinephrine by interacting with a specific presynaptic opiate receptor.

Differential effects of folic acid on water content, protein and microsomal 5'-phosphodiesterase activity of the rat kidney.

The effects of folic acid administration on the weight, protein, water content and microsomal 5'-phosphodiesterase of the rat kidney were determined, to elucidate the mechanisms contributing to the renal enlargement produced by this agent. Folic acid administered ip in single doses of 100-250 mg/kg caused dose-related increases in kidney weight, water and protein content within 24 hr. Time-course studies indicated that 250 mg folic acid/kg given ip produced a progressive elevation in renal water content from 2 to 72 hr. Smaller increases in whole-kidney protein were recorded 8, 24 and 72 hr after folic acid treatment. However, a biphasic response of microsomal 5'-phosphodiesterase was produced, inhibition at 16 hr being followed by stimulation (to 140% of control) at 72 hr. In vitro studies indicated that folic acid inhibits 5'-phosphodiesterase competitively, and the early inhibition of 5'-phosphodiesterase in vivo appears to be due to a direct effect of folic acid on the enzyme.

A procedure for dissociating Ayre scrape samples.

The dissociation of cervical cell suspensions after various chemical and enzymatic treatments was monitored by using the Centrifugal Cytology rotor to produce glutaraldehyde-fixed dispersions on conventional microscope slides and subsequent Pap staining. A special program was written in RPG II to record and analyze the results of the dissociation experiments in terms of white blood cells and the true cervical cells ("other cells"), and the degree of dissociation and recovery of both classes of cells. Since accurate differential counts on the untreated Ayre scrapes were difficult, the samples were syringed gently to break up the large or adventitious clumps. Cumulated results from control preparations indicate that the white blood cells and "other cells" are composed respectively of 92 and 63% single cells. The cells were further dissociated by: dissolving the cervical mucin sequentially with dithiothreitol and iodoacetic acid; depolymerizing the nucleohistone gel with ribonuclease; solubilizing the desmosomes with EDTA; removing the remaining cellular agglutinins with Varidase; and finally mechanical dispersion by hypertonic shock. The optimum procedure for dissociation involves the use of ribonuclease, dithiothreitol, iodoacetic acid EDTA, Varidase and sucrose shock. The white blood cells are now monodisperse and 81% of the "other cells" are found as single cells. If nuclear separation by two diameters is considered sufficient 98% of the "other cells" are single. The slide preparations are now sufficiently good that a scanning system is feasible.

Optimization of the binding of dissociated exfoliated cervico-vaginal cells to glass microscope slides.

In order to monitor the development of a cell dissociation technique, it was essential to utilize the Centrifugal Cytology rotor to produce glutaraldehyde-fixed even cellular dispersions. The Cytology rotor has been improved to insure rapid alignment with the centrifugal field during both acceleration and deceleration, and the fixative is now delivered to the surface of the slide. The dissociation of the cells results in a loss of their adhesion to glass slides. Three bonding agents were tested: (a) Poly-L-Lysine; (b) Mayer's albumin fixative; (c) positively charging the slides with a silicone coating. The results with 65% albumin-coated slides were clearly superior to the other two. The addition of a postfixation step of 95% ethanol/4% polyethylene glycol did not significantly affect the recovery of the cells, but did eliminate some unevenness in the Centrifugal Cytology preparations, flattened the cells and expedited the procedure.