George J. Trachte

Identification and characterization of the rabbit angiotensin II myocardial receptor.

The octapeptide, angiotensin II, elicits a positive inotropic response in myocardial tissue by activating slow calcium channels. Pharmacological studies suggest that the inotropic action of angiotensin II is receptor mediated. The current investigation was performed to characterize the binding of 123I-angiotensin II to the putative receptor in a plasma membrane-sarcoplasmic reticulum preparation of the rabbit left ventricle. In experiments performed at 18°C, steady state binding occurred at 45 minutes and saturable binding was 80–85% of the total binding. Analysis of the binding data indicated that the 125I-angiotensin II interacted with a single class of sites with a Kj = 4.5 ± 0.8 nM and exhibited a binding capacity of 53.5 ± 8 fmol/mg protein. The potency order for the competitive binding of analogues and antagonists of angiotensin II paralleled that observed for in vitro contractile force development in bioassay systems utilizing left atrial tissue. The binding of U:>I-angiotensin II was stimulated 2-fold in the presence of the divalent cations of calcium and magnesium (10 HIM). Guanine nucleotides modified the binding of 125I-angiotensin II to the rabbit myocardial particulate fraction. Guanine triphosphate and nonhydrolyzable analogues of guanine triphosphate increased the dissociation rate of the bound l2M-angiotensin II and decreased hormone binding to the receptor at equilibrium. In the absence of magnesium, guanine nucleotides had no effect on the dissociation rate of 125I-angiotensin II. I-I-Angiotensin II binding to a rabbit myocardial particulate fraction was found to have high affinity, to be saturable, reversible, specific, and modulated by guanine nucleotides. The characteristics of the binding site of l25I-angiotensin II indicate that it is the hormone receptor which mediates the physiological (inotropic) effects of angiotensin II in rabbit myocardial tissue

Potent constriction of cat coronary arteries by hydroperoxides of arachidonic acid and its blockade by anti-inflammatory agents

The omega-6 and omega-9 hydroperoxides of arachidonic acid caused dose-dependent constriction of cat coronary arteries in concentrations of 10(-8) to 10(-5) M. Their potency was comparable to that of prostaglandin (PG) E2, and PGF2 alpha and 100 times greater than that of arachidonic acid. The cyclooxygenase inhibitor, meclofenamate markedly reduced constriction caused by the hydroperoxides but potentiated constriction caused by the prostaglandins. The effects of the hydroperoxides were also reduced by indomethacin and dexamethasone but were unaffected by the thromboxane synthetase inhibitor imidazole. Since the hydroperoxides are not substrates for cyclooxygenase, it is suggested that they have a direct effect on the arteries which can be antagonized by anti-inflammatory drugs.

Prostaglandin production in response to angiotensin-(1-7) in rabbit isolated vasa deferentia

Angiotensin-(1-7) is a predominant metabolite of angiotensin I in brain tissue. Its neuromodulatory and prostaglandin (PG) synthesizing capabilities were investigated in the rabbit isolated vas deferens. This metabolite had no significant effect as a neuromodulator, however it increased PGE synthesis in the vasa deferentia with a potency equivalent to that of angiotensin II. The angiotensin-(1-7) has a unique spectrum of activity among the angiotensin peptides to selectively increase PG synthesis. It could be useful in defining the relevance of angiotensin-induced PG synthesis in various systems, particularly in neuronal tissue. Angiotensin-(1-7) potentially could be useful in defining angiotensin receptor subtypes, as well.

Angiotensin effects on vas deferens adrenergic and purinergic neurotransmission.

Angiotensin effects on purinergic and adrenergic neurotransmission in the rabbit vas deferens were examined. Both angiotensins inhibited the non-adrenergic, and potentiated the adrenergic, neurogenic contraction. Angiotensin III inhibited the non-adrenergic neurogenic contraction to a greater extent than angiotensin II at all concentrations tested (maximal inhibition being 42 +/- 4 vs. 17 +/- 3% for angiotensin II). Angiotensin II was more potent than angiotensin III at potentiating adrenergic neurotransmission. Neither peptide altered the postjunctional action of either putative neurotransmitter, ATP or norepinephrine. These results are inconsistent with the hypothesis that ATP and norepinephrine are released in constant ratios. Furthermore, the different pattern of angiotensin responses is consistent with the existence of at least two separate angiotensin receptors with markedly different affinities for angiotensin II and angiotensin III.

Immobilization increases bone prostaglandin E: Effect of acetylsalicylic acid on disuse osteoporosis studied in dogs

The effect of acetylsalicylic acid (aspirin) on bone mass and bone prostaglandin E (PGE) in immobilization osteoporosis was studied in 12 growing dogs using a unilateral hind limb cast-fixation model. Osteoporosis was induced by fiberglass-cast immobilization of the right hind limb for 4 weeks, with the left hind limb as a control. Six dogs received buffered aspirin at 25 mg/kg body weight per os every 8 hours; 6 dogs received no treatment. All the dogs were killed after 4 weeks, and bone samples were collected. Bone mineral content of the distal tibial metaphysis was measured by single-photon absorptiometry. In vitro release of PGE from the calcaneus, tibial cortical bone, tibial cancellous bone, and ilium were measured using a specific radioimmunoassay for PGE. Compared with the controls, the casted limb of untreated dogs had half the bone mass and a twofold increase in bone PGE. Aspirin treatment was associated with a 65 percent reduction in bone PGE and a 13 percent bone mass sparing effect. These results provide indirect evidence that PGE plays a role in immobilization osteoporosis.

Atrial natriuretic factor inhibits norepinephrine release in an adrenergic clonal cell line (PC12)

Evidence for a presynaptic neuromodulatory effect of atrial natriuretic factor (ANF) has been obtained in rat adrenal pheochromocytoma cells (PC12), a clonal line that differentiates into adrenergic neuron-like cells when treated with nerve growth factor. ANF had no effect on basal norepinephrine (NE) release. In contrast, ANF markedly and significantly inhibited carbachol-induced NE release in a concentration-dependent manner. This result is consistent with the hypothesis that ANF may be an inhibitory neuromodulator.

Both stimulatory and inhibitory effects of dietary 5-hydroxytryptophan and tyrosine are found on urinary excretion of serotonin and dopamine in a large human population

Amino acid precursors of dopamine and serotonin have been administered for decades to treat a variety of clinical conditions including depression, anxiety, insomnia, obesity, and a host of other illnesses. Dietary administration of these amino acids is designed to increase dopamine and serotonin levels within the body, particularly the brain. Convincing evidence exists that these precursors normally elevate dopamine and serotonin levels within critical brain tissues and other organs. However, their effects on urinary excretion of neurotransmitters are described in few studies and the results appear equivocal. The purpose of this study was to define, as precisely as possible, the influence of both 5-hydroxytryptophan (5-HTP) and tyrosine on urinary excretion of serotonin and dopamine in a large human population consuming both 5-HTP and tyrosine. Curiously, only 5-HTP exhibited a marginal stimulatory influence on urinary serotonin excretion when 5-HTP doses were compared to urinary serotonin excretion; however, a robust relationship was observed when alterations in 5-HTP dose were compared to alterations in urinary serotonin excretion in individual patients. The data indicate three statistically discernible components to 5-HTP responses, including inverse, direct, and no relationships between urinary serotonin excretion and 5-HTP doses. The response to tyrosine was more consistent but primarily yielded an unexpected reduction in urinary dopamine excretion. These data indicate that the urinary excretion pattern of neurotransmitters after consumption of their precursors is far more complex than previously appreciated. These data on urinary neurotransmitter excretion might be relevant to understanding the effects of the precursors in other organs.

The influence of prostaglandins on neurotransmission in the rabbit isolated vas deferens

Arachidonic acid and PGs of the D, E, F and I series were examined for influences on neurogenic contractions of the rabbit isolated vas deferens. This preparation exhibits two pharmacologically distinct contractions in response to electrical stimulation. All of the PGs tested inhibited the neurogenic contractions but the pattern of inhibition differed. PGE1 and PGI2 inhibited the adrenergic contractile phase more potently than the nonadrenergic, and PGF2 alpha exhibited the opposite selectivity. Arachidonic acid, PGE2 and PGD2 produced equipotent effects on both contractile phases, although PGE2 was the most potent in producing these effects. None of the PGs altered the concentration-response curve to norepinephrine. Contractile responses to ATP, a putative neurotransmitter, were inhibited by PGF2 alpha but not by the other PGs. These results suggest that the PG effects are predominantly prejunctional. The differing potencies of the PGs on the two neural components are consistent with the hypothesis that neurotransmitters in the vas deferens are released by distinct types of nerves.

Beneficial action of new angiotensin-converting enzyme inhibitor (SQ 14,225) in hemorrhagic shock in cats.

A new angiotensin-converting enzyme inhibitor (CEI), SQ 14,225, was infused at 0.5 mg/kg per hr, iv, into cats to determine its effect in hemorrhagic shock. Cats were bled to a mean arterial blood pressure (MABP) of 40 nun Hg for 150 minutes; this was followed by reinfusion and a 120-minute postoligemic observation period. Hemorrhagic shock and sham shock controls were given an infusion of the CEI or its vehicle (0.9% NaCl). The degree of converting enzyme inhibition was assessed by measuring pressor responses to angiotensin I and II and by radioimmunoassay determination of plasma angiotensin II concentrations. In vitro studies on cat papillary muscles and vascular smooth muscle strips revealed no direct inotropic or vasoactive effect of SQ 14,225. Nevertheless, hemorrhaged cats given the CEI demonstrated a significantly higher final arterial pressure than hemorrhaged cats given 0.9% NaCl (96 vs. 51 mm Hg) (P < 0.01), indicating a significant prolongation of circulatory stability which is closely related to survival. Circulating lysosomal hydrolase (i.e., cathepsin D) activity (3.5 vs. 11-fold increases) and total plasma proteolysis (25% vs. 100% increases) were significantly reduced in the shocked cats given the CEI compared to the untreated shocked animals. Formation of a myocardial depressant factor (MDF) also was significantly diminished by CEI treatment (26 vs. 62 U). These results indicate that CEI improved the hemodynamic and biochemical status of cats in hemorrhagic shock and suggest that blockade of angiotensin II formation may be beneficial in hemorrhagic shock. Abolition of other actions of converting enzymes (e.g., potentiation of bradykinin action or inhibition of proteolysis) may also be involved in the protective mechanisms.

C-type natriuretic peptide neuromodulates independently of guanylyl cyclase activation.

Of the four endogenous members of the natriuretic peptide family, only atrial natriuretic peptide has been demonstrated to have neuromodulatory effects. This study compares the neuromodulatory effects of atrial natriuretic peptide and a recently identified natriuretic peptide, C-type natriuretic peptide, in the rabbit isolated vas deferens. The ability of these peptides to alter cyclic nucleotide concentrations was assessed to determine the potential contribution of either cyclic AMP or cyclic GMP to the observed responses. The central hypothesis tested was that C-type natriuretic peptide modulates neurotransmission via an interaction with a guanylyl cyclase. C-type natriuretic peptide inhibited both purinergic and adrenergic neurotransmission in a concentration-dependent manner but failed to alter either cyclic GMP or cyclic AMP concentrations. Maximal inhibitory effects of C-type natriuretic peptide averaged 35 +/- 4% for purinergic and 49 +/- 7% for adrenergic neurotransmission. Atrial natriuretic peptide not only attenuated both purinergic and adrenergic neurotransmission but also increased cyclic GMP concentrations. C-type natriuretic peptide probably inhibited the release of the neurotransmitters because it failed to alter contractions to exogenously administered norepinephrine or ATP, the two putative neurotransmitters. These results suggest that the C-type natriuretic peptide receptor, guanylyl cyclase B, is not present in rabbit vas deferens and that C-type natriuretic peptide suppresses peripheral sympathetic neurotransmission independently of guanylyl cyclase activation.