Age-related changes of sodium-dependentd-[3H]aspartate and [3H]FK506 binding in rat brain

Summary We investigated age-related changes in excitatory amino acid transport sites and FK506 binding protein (FKBP) in 3-week-, and 6-, 12-, 18- and 24-month-old Fischer 344 rat brains using receptor autoradiography. Sodium-dependentd-[³H]aspartate and [³H]FK506 were used to label excitatory amino acid transport sites and immunophilin (FKBP), respectively. In immature rats (3-week-old), sodium-dependentd-[³H]aspartate binding was lower in the frontal cortex, parietal cortex, striatum, nucleus accumbens, whole hippocampus, thalamus and cerebellum as compared to adult animals (6-month-old), whereas [³H]FK506 binding was significantly lower only in the hippocampus, thalamus and cerebellum. [³H]FK506 binding exhibited no significant change in the brain regions examined during aging. However, sodium-dependentd-[³H]aspartate binding showed a conspicuous reduction in the substantia nigra in 18-month-old rats. Thereafter, a significant reduction in sodium-dependentd-[³H]aspartate binding was found in the thalamus, substantia nigra and cerebellum in 24-month-old rats. Other regions also showed about 10–25% reduction in sodium-dependentd-[³H]aspartate binding. The results indicate that excitatory amino acid transport sites are more susceptible to aging process than immunophilin. Further, our findings demonstrate the conspicuous differences in the developmental pattern between excitatory amino acid transport sites and immunophilin in immature rat brain.     

Differential modulation of angiotensin II and hypertonic saline-induced drinking by opioid receptor subtype antagonists in rats

Opioid modulation of ingestion includes general opioid antagonism of different forms of water intake, μ₂ receptor modulation of deprivation-induced water intake and δ₂ receptor modulation of saccharin intake. Water intake is stimulated by both central administration of angiotensin II (ANG II) and peripheral administration of a hypertonic saline solution; both responses are reduced by general opioid antagonists. The present study examined whether specific opioid receptor subtype antagonists would selectively alter each form of water intake in rats. Whereas systemic naltrexone (0.1–2.5 mg/kg, s.c.) reduced water intake induced by either peripheral ANGII (500 μg/kg, s.c.) or hypeptonic saline (3 ml/kg, 10%), intracerebroventricular (i.c.v.) naltrexone (1–50 μg) only inhibited central ANGII (20 ng)-induced hyperdipsia. Both forms of drinking were significantly and dose-dependently inhibited by the selective κ antagonist, nor-binaltorphamine (Nor-BNI, 1–20 μg). Whereas both forms of drinking were transiently reduced by the μ-selective antagonist, β-funaltrexamine (β-FNA, 1–20 μg), the μ₁ antagonist, naloxonazine (40 μg) stimulated drinking following hypertonic saline. The δ₁ antagonist, [d-Ala², Leu⁵, Cys⁶]-enkephalin (DALCE, 1–40 μg) significantly reduced drinking following ANGII, but not following hypertonic saline; the δ antagonist, naltrindole failed to exert significant effects. These data indicate that whereas κ opioid binding sites modulate hyperdipsia following hypertonic saline, μ₂, δ₁ and κ opioid binding sites modulate hyperdipsia following ANGII. The μ₁ opioid binding site may normally act to inhibit drinking following hypertonic saline.     

Regulation of vasopressin release in moderately severe essential hypertension

Vasopressin plasma concentrations have been measured in two groups of subjects, 13 moderate essential hypertensive patients without target organ damage and eight control normotensive subjects, before and after the assumption of the upright position, and intravenous infusions of hypotonic saline (0.45% NaCl, 0.25 ml kg^–1 min^–1 for 1 h) and hypertonic saline (100 mmol NaCl in 50 ml). Plasma vasopressin in recumbent baseline conditions was not significantly different in the two groups. Upright posture and hypertonic challenge augmented, while hypotonic saline reduced plasma vasopressin levels, which were not significantly different between the two groups. Plasma renin activity increased in the upright position, was reduced by administration of hypotonic saline and unaffected by hypertonic saline, with no differences between the hypertensives and normotensives. After hypertonic saline, urinary flow rate and urinary sodium excretion in the hypertensive group increased to values significantly (p < 0.05) higher than in normotensive subjects. In conclusion our study excludes significant alteration of vasopressin regulation in moderate uncomplicated hypertension. In hypertensives although the response of vasopressin to an osmotic load is preserved, the data suggest that the renal handling of the osmotic load may be altered.     

Potentiation of DOI-induced forward locomotion in rats by (−)-pindolol pretreatment

Summary The present experiments were undertaken in order to examine mechanisms of action for reported interactions between the -blocker (–)-pindolol and serotonergic agents. It was found that pretreatment with (–)-pindolol (2mg kg^–1 s.c.) potentiated the stereotyped forward locomotion induced by the 5-HT_2a/c receptor agonist DOI (0.125–1.0mg kg^–1 s.c.) in rats observed in an open-field arena. This (–)-pindolol/DOI-induced stereotyped forward locomotion was fully antagonized by the 5-HT_2a/c receptor antagonist ritanserin (2mg kg^–1 s.c.), suggesting that (–)-pindolol enhances serotonin release, resulting i.a. in postsynaptic 5-HT_2a/c receptor activation. This effect by (–)-pindolol is in all probability indirect since this compound lacks affinity for 5-HT_2a/c receptors, and could be explained by reported antagonism of inhibitory serotonergic somato-dendritic 5-HT_1a autoreceptors, although other possibilities related to 5-HT_1b receptors or -adrenoceptors can not be excluded at this time. Furthermore, (–)-pindolol treatment also enhanced 5-HTP-induced (12.5–100 mg kg^–1 i.p.) effects on spontaneous motor activity. These effects, however, were of smaller magnitude, and less consistent than those seen in combination with DOI.     

Depressed endothelium-dependent relaxation responses to acetylcholine and histamine in isolated human epigastric arteries from pre-eclamptic women

The vasorelaxant effects of acetylcholine (Ach) and histamine have been examined on 10^–7 m-noradrenaline (NA)-precontracted ring preparations of epigastric arteries from normotensive and pregnancy-induced hypertensive women. Contractile responses to 10^–7 m-NA were significantly (p<0.05) enhanced following removal of the endothelium. Both Ach and histamine (in the presence of H₁-receptor blockers) elicited methylene blue-sensitive concentration-dependent relaxations only in endothelium-intact rings. The relaxation responses to both agents were significantly attenuated in arterial rings from pre-eclamptic women. Endothelium-independent relaxation responses induced by sodium nitroprusside were comparable in arterial rings from both patient groups. The results suggest an impairment of endothelial function in pre-eclampsia.     

Neuropharmacological characterization of local ibogaine effects on dopamine release

Summary Local perfusion with ibogaine (10^–6M—10^–3M) via microdialysis probes in the nucleus accumbens or striatum of rats produced a biphasic dose-response effect on extracellular dopamine levels. Lower doses (10^–6M—10^–4M) produced a decrease while higher doses (5 × 10^–4M—10^–3M) produced an increase in dopamine levels. Dihydroxyphenylacetic acid (DOPAC) levels were not effected. Naloxone (10^–6M) and norbinaltorphimine (10^–6M—10^–5M) did not affect dopamine levels, but when co-administered with ibogaine (10^–4M) blocked the decrease in dopamine levels produced by ibogaine. Ibogaine (10^–3M) stimulation of dopamine levels in the striatum was calcium independent and not blocked by tetrodotoxin (10^–5M). Pretreatment with cocaine (15mg/kg), reserpine (5mg/kg) or alpha-methyl-paratyrosine (250mg/kg) given intraperitoneally significantly reduced ibogaine (10^–3M) stimulation of striatal dopamine levels. In striatal synaptosomes, both ibogaine and harmaline (10^–7—10^–4M) produced dose-dependent inhibition of [³H]-dopamine uptake. These findings suggest that ibogaine has both inhibitory and stimulatory effects on dopamine release at the level of the nerve terminal. It is suggested that the inhibitory effect is mediated by kappa opiate receptors while the stimulatory effect is mediated by interaction with the dopamine uptake transporter.     

The influence of ACTH and corticosterone on [H]GABA receptor binding in rat brain

Bilateral adrenalectomy (ADX) induces a significant, regionally selective, increase in GABA, but not cholinergic muscarinic orα₁-adrenergic, receptor binding in rat brain. The increase in GABA receptor binding in the midbrain occurs within 72 h of surgery, whereas that found in the corpus striatum becomes evident between 1 and 2 weeks later. These ADX-induced receptor changes are counteracted by the administration of corticosterone, a reversal which can occur within 24 h following a single administration of the steroid. Unlike ADX, hypophysectomy causes a significant reduction in [³H]GABA receptor binding in these two brain areas, an action that is not reversed by corticosterone treatment. Furthermore, systemic administration of either ACTH_1–39 or ACTH_4–10 in unoperated animals causes an increase in midbrain and striatal GABA receptor binding similar to that observed in ADX animals. The increase in [³H]GABA binding observed after ACTH administration appears to be due to the apearance of low affinity, high capacity binding sites not observed in untreated animals. ADX had no effect on high affinity GABA uptake, glutamic acid decar☐ylase or GABA content in the brain regions where receptor modifications were noted. These findings indicate that GABA receptor binding in rat brain can be modified by changes in the circulating levels of ACTH.     

Selective immunotoxin-induced cholinergic deafferentation alters blood flow distribution in the cerebral cortex

Adult rats received intracerebroventricular (i.c.v.) administration of either phosphate buffer (PBS) or 192 IgG-saporin (Toxin), 3.6 μg rat⁻¹, a cholinergic immunotoxin. Six to eight weeks later, the animals received a continuous intravenous (i.v.) infusion of either physostigmine (4.2 μg kg⁻¹ min⁻¹) or saline, followed by measurement of cerebral cortical blood flow (CBF) with the autoradiographic Iodo-¹⁴C-antipyrine methodology in four groups of animals: Toxin i.c.v.+saline i.v. (n=9), Toxin i.c.v.+physostigmine i.v. (n=6), PBS i.c.v.+saline i.v. (n=6) and PBS i.c.v.+physostigmine i.v. (n=6). Choline acetyltransferase activity (ChAT) was assessed with Fonnum's method in samples of cortical tissue adjacent to the sites of CBF measurement. ChAT decreased in all regions of the Toxin groups when compared to PBS (% decrease: hippocampus=93%, neocortex=80–84%, entorhinal-piriform cortex=42%, amygdala=28%). CBF decreased globally in Toxin+SAL, most severely in posterior parietal and temporal regions (24–40% decrease from PBS+saline). Physostigmine enhanced CBF predominantly in these same areas both in PBS and Toxin animals although to a lesser extent in the latter. Our results demonstrate the importance of cholinergic mechanisms in the control of CBF. The similarity between the topography of CBF decrease following administration of the immunotoxin to that observed in Alzheimer's disease suggests that the CBF pattern observed in this disease may be the result of cholinergic deafferentation.     

Effect of cholinergic blockade on heart rate,blood pressure and plasma catecholamine responses to mental stress in normal subjects

The effects of cholinergic blockade on haemodynamic reactivity to standardized mental stress has been studied in nine normotensive males during infusion of atropine (bolus dose 10 µg × kg^–1 followed by a constant-rate infusion of 0.02 µg × kg^–1 × min^–1) or placebo given in a randomized order on two different days. Partial cholinergic blockade increased resting heart rate by 25–30 beats per minute. The magnitude of the heart rate response to stress (reactivity) however was unaffected by the atropine infusion. Also, in four subjects who received a higher dose of atropine (approximately 1.8–1.9 mg), heart rate responses to stress were the same as during placebo infusion. Cholinergic blockade was associated with a small but prolonged increase in diastolic blood pressure. These findings suggest that parasympathetic withdrawal does not contribute to the tachycardia caused by mental arithmetic, and that the pattern of neurogenic activation may differ from that elicited during a classic defence-alarm reaction and by somatomotor activation.     

Differential effects of prostanoid synthesis inhibitors on cicletanine-induced relaxation of isolated human epigastric arteries

We have examined the influence of prostanoid synthesis inhibitors on the relaxation responses induced by cicletanine in ring preparations of isolated human epigastric arteries following precontraction induced by 10^–7 M noradrenaline. Cicletanine caused concentration-dependent relaxations, uninfluenced by the cyclooxygenase inhibitors, meclofenamate (1, 10 µM) and indomethacin (1, 10 µM) but significantly (p < 0.05) attenuated by the specific prostacyclin synthesis inhibitor, tranylcypromine (0.1–10 µM)(n > 6 in each). In contrast, cromakalim-induced relaxations were significantly attenuated by indomethacin, meclofenamate and tranylcypromine. The tranylcypromine-sensitive cicletanineinduced relaxation suggests, at least in part, the involvement of prostacyclin in the vasodilator action of cicletanine.     

Effects of dopamine on cyclic AMP concentration in the anterior pituitary glandin vitro

Summary Effects of different concentrations of dopamine on the cyclic AMP concentration in the rat anterior pituitary gland were investigated in vitro. Low concentrations of dopamine (10^–9–10^–8 mol/l) were found to decrease, whereas the high concentration (10^–5 mol/l) increased the cyclic AMP concentration in pituitaries collected from ovariectomized and estradiol-treated females. In contrast, dopamine had no effect on the anterior pituitary cAMP concentration when pituitaries were collected from ovariectomized rats which had not received estrogen replacement. These data show that the action of dopamine on the anterior pituitary cAMP largely depends on the dopamine concentration and the hormonal state of animals.This paper was supported by the Polish Academy of Sciences Grant No. 10.4.2.01.5.5.     

[Leu,Pro]Neuropeptide Y (NPY), but not NPY 20–36, produces discriminative stimulus effects similar to NPY and induces food intake

Rats were trained to discriminate between an intracerebroventricular injection of 1.15 nmol of Neuropeptide Y (NPY) and a sham injection. Rats rapidly learned to press the appropriate lever during training. NPY's discriminative stimulus effects were compared to those of saline, and 1.15–3.45 nmol [Leu³¹,Pro³⁴]NPY, a Y₁ receptor agonist and NPY 20–36, Y₂ receptor agonist. [Leu³¹,Pro³⁴]NPY resulted in NPY-appropriate responding, whereas saline and NPY 20–36 did not. [Leu³¹,Pro³⁴]NPY also increased food intake, but NPY 20–36 did not. This suggests that NPY's discriminative stimulus and orexigenic effects involve the Y₁, but not the Y₂, receptor.     

Regional impairment of protein synthesis following brief cerebral ischemia in the gerbil

Summary Regional cerebral protein synthesis following brief ischemia was investigated in the Mongolian gerbil, utilizing l-[methyl-¹⁴C]methionine autoradiography. Transient ischemia was induced for 1,2 or 3 min. At various recirculation periods up to 48 h, animals received a single dose of l-[methyl-¹⁴C]-methionine and then were terminated 35 min later. Sham-operated animals showed a normal pattern of amino acid incorporation into the proteins of the brain. Following 1-min ischemia, the pattern of protein synthesis was similar to that in the sham-operated gerbils. Ischemia for 2 min, however, caused marked inhibition of protein synthesis in the neocortex, striatum, hippocampal CA1 sector and the thalamus at 1 h of recirculation. Extensive recovery of protein synthesis was found in the neocortex, the striatum, the hippocampal CA1 sector and the thalamus at 5–24 h of recirculation, but, a slight inhibition was detectable in the hippocampal CA1 sector in one of six animals. This inhibition had fully recovered at 48 h of recirculation. Following 3-min ischemia, severe impairment of protein synthesis was found in the neocortex, striatum, the whole hippocampus and the thalamus. After 5–24 h of recirculation, the protein synthesis in these regions had gradually recovered, except that complete lack of amino acid incorporation was seen in the hippocampal CA1 subfield. This impairment of protein synthesis in the hippocampal CA1 sector was not recovered at 48h of recirculation. Morphological study indicated that 2-min ischemia did not produce any significant neuronal damage in the brain, whereas gerbils subjected to 3-min ischemia revealed a mild neuronal damage in the hippocampal CA1 sector. The present study indicates that even non-lethal ischemia can produce a severe inhibition of protein synthesis in the selectively vulnerable regions during the early stage of recirculation.     

Postnatal ethanol exposure blunts upregulation of GABAA receptor currents in Purkinje neurons

Recently, we found that early postnatal ethanol exposure inhibits the maturation of GABA_A receptors (GABA_ARs) in developing medial septum/diagonal band (MS/DB) neurons, suggesting that these receptors may represent a target for ethanol related to fetal alcohol syndrome (FAS). To determine whether GABA_ARs on other neurons are also sensitive to a postnatal ethanol insult, postnatal day (PD) 4–9, rat pups were artificially reared and exposed to ethanol (4.5 g kg⁻¹ day⁻¹, 10.2% v/v). The pharmacological profile of acutely dissociated cerebellar Purkinje cell GABA_ARs from untreated, artificially reared controls and ethanol-treated animals was examined with conventional whole-cell patch clamp recordings during PD 12–16 (juveniles) and PD 25–35 (young adults). For untreated animals, GABA (0.3–100 μM) consistently induced inward Cl⁻ currents in a concentration-dependent manner showing an age-related increase in maximum response without change in EC₅₀ or slope value. Acute ethanol (100 mM) consistently inhibited 3 μM GABA currents (10–20%); positive modulators, pentobarbital (10 μM), midazolam (1 μM) and loreclezole (10 μM), consistently potentiated; the negative modulator, Zn²⁺ (30 μM), inhibited GABA currents across both juvenile and young adult groups. Loreclezole potentiation increased while Zn²⁺ inhibition decreased with age in untreated Purkinje neurons. Postnatal ethanol exposure (PD 4–9) decreased GABA_AR maximum current density in young adult Purkinje cells but not in juvenile neurons. However, sensitivity to allosteric modulators did not change after ethanol. These data are consistent with the hypothesis that postnatal ethanol exposure during the brain growth spurt can disturb GABA_AR development across the brain, although the mechanism(s) underlying this action remains to be determined.     

Comparison of GABA analogues at the crayfish stretch receptor neurone

The conductance increase induced by GABA and structurally related compounds has been measured in voltage clamped stretch receptor neurones of crayfish. GABA induced only at 10⁻³ M a rapid conductance increase. The response to lower concentrations between 10⁻⁶ and 10⁻⁴ M developed slowly (20–60 min). The postsynaptic conductance increase induced by repetitive application of the same GABA concentration was progressively enhanced in the speed and magnitude. In the presence of nipecotic acid or in Na⁺-free Ringer solutions, the response to all GABA concentrations was instantaneous and constant for each concentration. Muscimol between 10⁻⁶ and 10⁻³ M caused instantaneous dose-dependent conductance increases. These results suggest the presence of a saturable GABA uptake system limiting the access of bath applied GABA, but not of muscimol, to postsynaptic receptor sites.     

The yohimbine-induced anticonflict effect in the rat,part II. Neurochemical findings

Summary In a companion paper the ₂-adrenoceptor antagonist yohimbine was found to produce a dose-dependent anticonflict effect in a modified Vogel's conflict test. The behavioral data further indicated that noradrenergic and serotonergic neurons as well as the benzodiazepine (BDZ) receptor may be involved in the anticonflict effect of yohimbine. In the present study the effects on rat brain monoamine neurochemistry and GABA _a /BDZ receptor function (³⁶Cl^–-uptake in corticohippocampal synaptoneurosomes) of a maximally anticonflict producing dose of yohimbine (4.0 mg/kg, i.p.) were studied. The levels of rat brain catecholamines and indoleamines were measured ex vivo using high performance liquid chromatography with electrochemical detection (HPLC-ED). Yohimbine decreased noradrenaline levels both in the hippocampus and the hemispheres but instead increased DOPAC levels in these brain regions as well as in the limbic forebrain. Yohimbine also markedly enhanced DOPA accumulation in the hippocampus and the hemispheres after inhibition of 1-aromatic amino acid decarboxylase by means of NSD 1015, whereas in the limbic system only a modest increase was obtained. The yohimbine-induced effects on the catecholamine synthesis rate were largely abolished in animals severely depleted of NA by means of 6-hydroxy-dopamine (6-OH-DA) pretreatment. Yohimbine decreased both the 5-HIAA/5-HT quotient (an indicator of 5-HT turnover) and 5-HTP accumulation after NSD 1015 in the hemispheres, whereas in the hippocampus and the limbic system only 5-HTP accumulation was decreased. The yohimbine-induced effect on the indoleamine synthesis rate was not influenced by 6-OH-DA pretreatment, whereas this effect and that on the catecholamine synthesis rate were both abolished by reserpine pretreatment. Neither in vivo nor in vitro administration of yohimbine significantly altered baseline or GABA-induced accumulation of³⁶Cl^– in corticohippocampal synaptoneurosomes. In conclusion, the present study provides neurochemical support for the suggestion that yohimbine may exert its anticonflict effect in a modified Vogel's conflict test by increasing and decreasing NA and 5-HT neurotransmission, respectively, whereas no evidence was obtained for a direct interaction of yohimbine with GABA _a /BDZ receptor function.     

Naltrexone accelerates healing without compromise of adhesion complexes in normal and diabetic corneal epithelium

Naltrexone (NTX) is an opioid antagonist that accelerates wound healing of corneal epithelium in normal and diabetic animals. Junctional complexes (hemidesmosomes) are important in establishing adhesion of the corneal epithelium to the stroma. This study was designed to examine whether NTX, at a concentration that enhances corneal re-epithelialization, influences the appearance and number of hemidesmosomes in Normal, diabetic (DB) (hyperglycemic), and DB animals receiving insulin (DB-IN) (normoglycemic), and treated topically with NTX (10⁻⁴ M) or sterile vehicle (SV) for 7 days following abrasion. Electron microscopic analysis of the peripheral cornea 2 weeks after removal of the epithelium indicated hemidesmosomes that could be classified into four sectional profiles. No differences were detected in either the structure or the number of junctional complexes in the cornea between Normal, DB, or DB-IN groups receiving vehicle or treated with NTX. Moreover, the fine structure of the basal and suprabasal layers of the corneal epithelium in all groups – including those treated with NTX – were comparable. These results indicate that topical application of NTX accelerates diabetic corneal epithelial healing without causing morphologic abnormalities in the reassembly of adhesion structures. Furthermore, controlled and uncontrolled diabetes for up to 3 months does not affect corneal adhesion complexes when compared to normal corneas. Thus, recurrent erosion following abrasion of the diabetic cornea, with preservation of the basal lamina, cannot be explained by structural abnormalities in the reformation of the epithelial adhesion complex.     

The co-expression of VR1 and VRL-1 in the rat vagal sensory ganglia

Immunohistochemistry for two nociceptive transducers, the vanilloid receptor 1 (VR1) and vanilloid receptor 1-like receptor (VRL-1), was performed on the vagal sensory ganglia. In the jugular ganglion, VR1-immunoreactive (IR) neurons were small to medium-sized (range 49.7–1125.6 μm², mean±S.D. 407.7±219.7 μm²), whereas VRL-1-IR neurons were medium-sized to large (range 223.6–1341.1 μm², mean±S.D. 584.3±253.5 μm²). In the nodose ganglion, VR1- and VRL-1-IR neurons were mostly small to medium-sized (VR1: range 148.5–1464.4 μm², mean±S.D. 554.3±207.4 μm²; VRL-1: range 161.7–1166.2 μm², mean±S.D. 541.9±186.2 μm²). The double immunofluorescence method revealed that co-expression of VR1-immunoreactivity among VRL-1-IR neurons was more abundant in the nodose ganglion (63%) than in the jugular ganglion (4%). The present study suggests that co-expression of VR1 and VRL-1 may be more common in visceral sensory neurons than in somatic sensory neurons.     

Angiotensin-(1–7) causes endothelium-dependent relaxation in canine middle cerebral artery

The heptapeptide, angiotensin-(1–7), is an active member of the renin–angiotensin system. The present study was designed to characterize the role of endothelium in relaxations of large cerebral arteries to angiotensin-(1–7). Rings of canine middle cerebral arteries were suspended in organ chambers for isometric force recording. The levels of cyclic guanosine 3′,5′-monophosphate (cGMP) were assessed by radioimmunoassay. During contraction to uridine 5′-triphosphate (UTP, 3×10⁻⁶ to 10⁻⁵ mol/l), angiotensin-(1–7) (10⁻⁹ to 3×10⁻⁵ mol/l) caused concentration-dependent relaxations in arteries with endothelium, but not in endothelium-denuded vessels. Angiotensin-(1–7) significantly increased formation of cGMP. Nitric oxide synthase inhibitor, N-ω-nitro- -arginine methyl ester ( -NAME, 3×10⁻⁴ mol/l), and selective soluble guanylate cyclase inhibitor, 1 H-[1,2,4]oxadiazolo[4,3-a]quinozalin-1-one (ODQ, 3×10⁻⁶ mol/l), abolished angiotensin-(1–7)-induced relaxations. Angiotensin receptor antagonists, losartan (10⁻⁵ mol/l), PD 123 319 (10⁻⁵ mol/l), [Sar¹,Thr⁸]-angiotensin II (10⁻⁵ mol/l) [Sar¹,Val⁵,Ala⁸]-angiotensin II (10⁻⁵ mol/l) or [7- -Ala]-angiotensin 1–7 (10⁻⁶ mol/l) did not affect these relaxations. However, angiotensin-converting enzyme inhibitor, captopril (10⁻⁵ mol/l) augmented relaxations to angiotensin-(1–7). Finally, bradykinin B₂ receptor antagonist, [ -Arg⁰,Hyp³,Thi⁵, -Tic⁷,Oic⁸]-bradykinin (HOE 140, 5×10⁻⁸ mol/l) significantly reduced the effect of angiotensin-(1–7), while bradykinin B₁ receptor antagonist, des-Arg⁹, [Leu⁸]-bradykinin (6×10⁻⁹ mol/l) did not influence the vascular response to the heptapeptide. These findings indicate that (1) angiotensin-(1–7) produces relaxation of canine middle cerebral arteries by the release of nitric oxide from endothelial cells, (2) angiotensin receptors do not mediate endothelium-dependent relaxations to the heptapeptide, and (3) this effect appears to be dependent on activation of local production of kinins. Our studies support the concept that angiotensin-(1–7), as a natural vasodilator hormone, may counterbalance the hemodynamic actions of angiotensin II.     

Ryanodine receptor-mediated [Ca]i release in glomus cells is independent of natural stimuli and does not participate in the chemosensory responses of the rat carotid body

The hypothesis that intracellular calcium ([Ca²⁺]_i) release in glomus cells via ryanodine receptor (RyR) activation by caffeine may be independent of natural stimuli and chemosensory discharge was tested in the rat carotid body (CB). CB type I cells were isolated, plated and preloaded with calcium-sensitive fluorescent probe, Indo-1AM. With the increase of caffeine dose (0–50 mM) cytosolic calcium ([Ca²⁺]_c) increased from 85±15 nM to 1933±190 nM (n=6) at normoxia (P ₂=125–130 Torr, P ₂=25–30 Torr, pH 7.30–7.35). Hypoxia (P ₂=10–15 Torr) increased and hypocapnia (P ₂=7–9 Torr) decreased the cytoplasmic calcium [Ca²⁺]_c levels, independent of caffeine. Caffeine-related [Ca²⁺]_c increase was the same in the presence and the absence of extracellular calcium ([Ca²⁺]_o), indicating the source of Ca²⁺ ions is the cellular store. Permeabilization of the cell membrane with saponin (25 μg/ml) retained the caffeine response. Additional treatment of the cells with 50 μM ryanodine (an inhibitor of the caffeine-activated RyR site) abolished caffeine-stimulated response. In vitro CB chemosensory (carotid sinus nerve, CSN) responses to hypoxia (P ₂=35–40 Torr) were not altered by caffeine. These results suggest that [Ca²⁺]_i stores in CB cells, mobilized by RyR activation, do not participate in the CSN responses to natural stimuli.