Effect of adaptation to cold on the alpha1- and beta-adrenergic reactions of arterial vessels of the small intestine in rabbits

Changes in major paraments of alpha 1- and beta-adrenergic responses (EC50 and Pm) were studied in the intestine arterial blood vessels of rabbits adapted to cold for 1-30 days (daily cold exposures for 6 hours at -10 degrees C). It was shown that responses to phenylephrine, noradrenaline, adrenaline (alpha 1-agonists), isopropylnoradrenaline (beta-agonist) corresponded to the equation p = (Pm.An)/(EC50n + An) with n = 1 and n = 2, respectively. Adaptation to cold induced radically different changes in the major parameters of alpha- and beta-adrenergic responses. In the alpha-adrenergic responses, the parameters EC50 and Pm changed reciprocally. In the beta-adrenergic response, only Pm value changed while EC50 did not differ from the control over the entire period of adaptation to cold. The pronounced differences from the control gradually decreased within 1-30 days of adaptation.     

Changes of alpha1-adrenergic and muscarinic cholinergic responses of arterial blood pressure in the rabbit during adaptation to cold

Changes in major parameters of alpha1-adrenergic and muscarinic cholinergic responses of arterial blood pressure (EC50 and P(m)) were studied in rabbits adapted to cold for 1-30 days (daily cold exposures for 6 hours at -10 degrees C). It was shown that responses to noradrenaline, adrenaline (agonists of alpha1-adrenoceptors) and acetylcholine (agonist of muscarinic cholinoreceptors) could be expressed by equation p = (Pm- A(n))/(EC50(n) + A(n)) with n = 1. Adaptation to cold induced radically different changes in the major parameters of adrenergic and cholinergic responses. In the noradrenergic responses, the parameters EC50 and Pm gradually decreased within 1-30 days of adaptation. In the adrenaline responses, Pm increased from 1 to 30 days of adaptation, EC50 decreased to 10 day and increased on the 30th day. In the muscarinic cholinergic response, the Pm value decreased from 1 and 10 days but returned to control values on the 30th day. IC5O did not differ from the control 1 day, decreased to 10 days and increased on the 30th day.     

Quantitative analysis of ligand-receptor interactions in physiological experiments

In isolated smooth muscles of the sea cucumber, the rat intestine, vas deferens and portal vein, and in chick embryonic amnion, contractile responses of smooth muscles to transmitters and their agonists were described with two equations: p = (Pm.A(n))/(EC50n + A(n)) [7] or p = [(Pm1.An1)/(EC50(1)n1 + An1)] + [(Pm2.An2)/(EC50(2)n2 + An2)] [8]. The findings reveal a possibility of ligand-receptor interaction according to several models: a single receptor pool with n = 1 or n not equal to 1; two receptor pools in the same effector system with n1 = n2 or n1 not equal to n2.     

Electrophysiological evidence for the coexistence of alpha1 and alpha6 subunits in a single functional GABA(A) receptor

The subunit combinations alpha1beta2gamma2, alpha6beta2gamma2, and alpha1alpha6beta2gamma2 of the GABA(A) receptor were functionally expressed in Xenopus oocytes. The properties of the resulting ion currents were characterized by using electrophysiological techniques. The concentration-response curve of the channel agonist GABA for alpha1alpha6beta2gamma2 showed a single apparent component characterized by an EC(50) of 107 +/- 26 microM (n = 4). It was different from the one for alpha1beta2gamma2, which had an EC(50) of 41 +/- 9 microM (n = 4), that for alpha6beta2gamma2, with an EC(50) of 6.7 +/- 1.9 microM (n = 5), and those for alpha1beta2 and alpha1alpha6beta2. There was no appreciable functional expression of alpha6beta2. Allosteric responses of alpha1alpha6beta2gamma2 to diazepam were intermediate to those of alpha1beta2gamma2 and alpha6beta2gamma2, and allosteric responses to flumazenil were comparable to the ones for alpha1beta2gamma2. The inhibition by furosemide of the currents elicited by GABA in alpha1alpha6beta2gamma2 [IC(50) = 298 +/- 116 microM (n = 7), assuming only one component] was not identical with inhibition of alpha6beta2gamma2 (IC(50) = 38 +/- 2 microM, n = 4), alpha1beta2gamma2 (IC(50) = 5,610 +/- 910 microM, n = 5), or a mixture of these components (assuming two components). These findings indicate unambiguously the formation of functional GABA(A) receptors containing two different alpha subunits, alpha1 and alpha6, with properties different from those of alpha1beta2gamma2 and alpha6beta2gamma2. Furthermore, we provide evidence for the facts that in the Xenopus oocyte (a) the formation of the different receptor types depends on the relative abundance of cRNAs coding for the different receptor subunits and (b) that functional dual subunit combinations alphabeta do not form in the presence of cRNA coding for the gamma subunit.     

Essential role of calcium influx in the adrenergic regulation of cAMP and cGMP in rat pinealocytes

The role of Ca2+ in the adrenergic stimulation of pinealocyte cAMP and cGMP was investigated. In this tissue alpha 1-adrenoceptor activation, which by itself is without effect, potentiates beta 1-adrenergic stimulation of cAMP and cGMP 30- to 100-fold. The present results indicate that chelation of extracellular Ca2+ with EGTA or inhibition of Ca2+ influx with inorganic Ca2+ channel blockers (La3+, Co2+, Mn2+) markedly reduces the cyclic nucleotide response to norepinephrine, a mixed alpha 1- and beta-adrenergic agonist, but not to isoproterenol, a beta-adrenergic agonist. In addition, the potentiating effects of alpha 1-adrenergic agonists were mimicked by agents which elevate cytosolic Ca2+, including K+ (EC50 = 2 X 10(-2) M), ouabain (EC50 = 2 X 10(-6) M), ionomycin (EC50 = 3 X 10(-6) M), and A23187 (EC50 = 2 X 10(-6) M); each potentiated the effects of beta-adrenergic stimulation but had no effect alone. Together these results indicate that an alpha 1-adrenoceptor-stimulated Ca2+ influx is essential for norepinephrine to increase pinealocyte cAMP and cGMP.     

EC3, a novel heterodimeric disintegrin from Echis carinatus venom, inhibits alpha4 and alpha5 integrins in an RGD-independent manner

EC3, a heterodimeric disintegrin (Mr = 14,762) isolated from Echis carinatus venom is a potent antagonist of alpha4 integrins. Two subunits called EC3A and EC3B were isolated from reduced and alkylated EC3 by reverse-phase high performance liquid chromatography. Each subunit contained 67 residues, including 10 cysteines, and displayed a high degree of homology to each other and to other disintegrins. EC3 inhibited adhesion of cells expressing alpha4beta1 and alpha4beta7 integrins to natural ligands vascular cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1 (MadCAM-1) with IC50 = 6-30 nM, adhesion of K562 cells (alpha5beta1) to fibronectin with IC50 = 150 nM, and adhesion of alphaIIbbeta3 Chinese hamster ovary cells to fibrinogen with IC50 = 500 nM; it did not inhibit adhesion of alphavbeta3 Chinese hamster ovary cells to vitronectin. Ethylpyridylethylated EC3B inhibited adhesion of Jurkat cells to immobilized VCAM-1 (IC50 = 6 microM), whereas EC3A was inactive in this system. The MLDG motif appeared to be essential for activity of EC3B. Linear MLDG peptide inhibited the adhesion of Jurkat to VCAM-1 in a dose-dependent manner (IC50 = 4 mM), whereas RGDS peptide was not active at the same concentration. MLDG partially inhibited adhesion of K562 cells to fibronectin (5-10 mM) in contrast to RGDS peptide (IC50 = 3 mM), inhibiting completely at 10 mM.     

Roles of alpha 1- and beta-adrenergic receptors in adrenergic responsiveness of liver cells formed after partial hepatectomy

The adrenergic receptor involved in the action of epinephrine changed dramatically during the process of active proliferation which follows partial hepatectomy. In control or sham-operated animals, the stimulation of glycogenolysis, gluconeogenesis and ureogenesis by epinephrine was mediated through alpha 1-adrenergic receptors. In contrast, in hepatocytes obtained from animals partially hepatectomized 3 days before experimentation, the receptor involved in the stimulation of these metabolic pathways by epinephrine was of the beta-adrenergic type. Interestingly, the adrenergic receptor involved in the metabolic actions of epinephrine, in hepatocytes from rats partially hepatectomized 7 days before experimentation was again of the alpha 1-subtype. Thus, it appears that during the process of liver regeneration which follows partial hepatectomy there is a transition in the type of adrenergic receptor involved in the hepatic actions of catecholamines from beta in the initial stages to later alpha 1. A similar transition seems to occur as the animal ages. Cyclic AMP accumulation in response to beta-adrenergic stimulation was significantly enhanced in hepatocytes obtained from rats partially hepatectomized 3 days before the experiment, as compared to control hepatocytes or cells obtained from animals operated 7 days before experimentation. This enhanced beta-adrenergic sensitivity is probably related to the increased number of beta-adrenergic receptors observed at this stage. However, a clear dissociation between cyclic AMP levels and metabolic effects was evidenced when the different conditions were compared. The number and affinity (for epinephrine or prazosin) of alpha 1-adrenergic receptors did not change at any stage of the process, which indicates that the markedly diminished alpha 1-adrenergic sensitivity observed in hepatocytes obtained from rats partially hepatectomized 3 days before experimentation is probably due to defective generation or intracellular processing of the alpha 1-adrenergic signal, rather than to changes at the receptor level.     

Ontogeny of alpha 1- and beta-adrenergic receptors in rat lung

The binding characteristics of the alpha 1-selective adrenergic ligand [3H]-prazosin were determined in particulate membranes of rat lung from day 18 of gestation to adulthood. Specific binding was present at all ages studied, was reversible and inhibition of specific binding by agonists followed the order of potency: (-)-epinephrine = (-)-norepinephrine much greater than (-)-isoproterenol greater than (+)-norepinephrine. Inhibition by antagonists followed the order of potency: prazosin greater than WB4101, much greater than yohimbine. Binding capacity increased during the neonatal period from 52 +/- 9 fmoles x mg-1 protein in lung preparations on day 18 of a 21 day gestation increasing to 105 +/- 4 fmoles x mg-1 protein (mean +/- SE) by postnatal day 15. Binding activity decreased thereafter, reaching adult levels by 28 days of postnatal age, 62 +/- 3 fmoles x mg-1 protein. This pattern of alpha 1-adrenergic receptor density was distinct from that of beta-adrenergic receptors identified in rat lung membrane with the beta- adrenergic antagonist, (-)-[3H]dihydroalprenolol ((-)-[3H]DHA). (-)-[3H]DHA binding increased dramatically during this same time period, from 46 +/- 4 fmoles x mg-1 protein on day 18 of gestation to 496 +/- 44 fmoles x mg-1 protein in the adult lung. Affinity for [3H]-prazosin and (-)-[3H]DHA did not change with age. Pulmonary alpha 1-adrenergic receptors are present as early as 18 days of gestation in the rat and alpha 1-adrenergic receptor density is maximal by 15 days of postnatal age. The timing of the changes in alpha 1-adrenergic receptors correlates with the timing of increased sympathetic innervation of the developing rat lung and is distinct from that of beta-adrenergic receptor sites.     

Suppression of prostaglandin F-2 alpha release and delay of luteolysis after active immunization against oxytocin in the goat

Active immunization against oxytocin significantly prolonged the oestrous cycle in 3 out of 4 goats; the mean (+/- s.e.m.) cycle length was 29.1 +/- 1.7 days (n = 12) compared to 19.4 +/- 0.6 days (n = 9) in control animals. During Days 10-21 of the cycle in the 3 responsive goats, peripheral plasma concentrations of progesterone and oxytocin were steady and those of 13,14-dihydro-15-keto-prostaglandin F-2 alpha were very low (50-100 pg X ml-1) with no marked pulsatile activity. The major effect of immunization would appear to be suppression of the synthesis of the uterine luteolysin PGF-2 alpha, thus confirming that endogenous oxytocin has a facilitatory role in luteolysis via prostaglandin production.     

Synchronisation of oestrus in dairy cows using prostaglandin F2alpha,gonadotrophin-releasing hormone,and oestradiol cypionate

An oestrous synchronisation protocol was developed for use in lactating dairy cows using PGF(2alpha), GnRH, and oestradiol cypionate (ECP). In experiment 1, lactating dairy cows received two injections of PGF(2alpha) (on days 0 and 11) (PP; n=10) or two injections of PGF(2alpha) (days 0 and 11) and 100 microg of GnRH on day 3 (PGP; n=10). In experiment 2, cows were treated with PGP (n=7), or PGP and 1 mg of ECP at the same time (PGPE(0); n=7) or 1 day after the second PGF(2alpha) injection (PGPE(1); n=7). In experiment 3, 101 lactating dairy cows in a commercial herd were assigned to one of three treatments; PP, PGP, or PGPE(1). Follicular growth was measured by ultrasound in experiments 1 and 2. Every cow (experiments 1, 2, and 3) was blood sampled at selected intervals for progesterone and oestradiol assays and inseminated at oestrus. In experiment 1, a higher percentage of GnRH-treated cows ovulated after the first PGF(2alpha) injection (90% versus 50%; P<0.05). The GnRH-treated cows tended to have a larger dominant follicle present at the time of the second PGF(2alpha) injection (16.5+/-0.5 mm versus 15.0+/-0.7 mm; P<0.10). The percentage of cows that ovulated after the second PGF(2alpha) injection was similar (60%). In experiment 2, cows treated with ECP had higher peak preovulatory concentrations of oestradiol in plasma (6.99+/-0.63 versus 3.63+/-0.63; P<0.01) following the second PGF(2alpha) injection and a higher percentage ovulated (86% versus 43%; P<0.05). A higher percentage of PGPE(1)-treated cows in experiment 3 were observed in standing oestrus and ovulated after the second PGF(2alpha) injection (standing oestrus, 26.4, 34.3, and 62.6%, P<0.01; ovulated, 56, 63, and 78%, P<0.05; PP, PGP, and PGPE(1), respectively). In conclusion, the PGP protocol increased the number of cows that ovulated after the first PGF(2alpha) injection and produced a more mature dominant follicle at the time of the second PGF(2alpha) injection. Adding ECP to PGP (PGPE(1)) enhanced the expression of oestrus and increased ovulation percentage. The combination of PGP and ECP is potentially a new method to routinely synchronise oestrus and ovulation in dairy cows.     

Selective alpha2-adrenergic properties of dexmedetomidine over clonidine in the human forearm.

We tested the hypothesis that dexmedetomidine (Dex) has greater alpha(2)- vs. alpha(1) selectivity than clonidine and causes more alpha(2)-selective vasoconstriction in the human forearm. After local beta-adrenergic blockade with propranolol, forearm blood flow (plethysmography) responses to brachial artery administration of Dex, clonidine, and phenylephrine (alpha(1)-agonist) were determined in healthy young adults before and after alpha(2)-blockade with yohimbine (n = 10) or alpha(1)-blockade with prazosin (n = 9). Yohimbine had no effect on phenylephrine-mediated vasoconstriction but blunted Dex-mediated vasoconstriction (mean +/- SE: -41 +/- 5 vs. -11 +/- 2%; before vs. after yohimbine) more than clonidine-mediated vasoconstriction (-39 +/- 5 vs. -28 +/- 4%; before vs. after yohimbine) (P < 0.02). Prazosin blunted phenylephrine-mediated vasoconstriction (-39 +/- 4 vs. -8 +/- 2%; before vs. after prazosin) but had similar effects on both Dex- (-30 +/- 4 vs. -39 +/- 6%; before vs. after prazosin) and clonidine-mediated vasoconstriction (-29 +/- 3 vs. -41 +/- 7%; before vs. after prazosin) (P > 0.7). Both Dex and clonidine reduced deep forearm venous norepinephrine concentrations to a similar extent (-59 +/- 12 vs. -55 +/- 10 pg/ml; Dex vs. clonidine, P > 0.6); this effect was abolished by yohimbine and blunted by prazosin. These results suggest that Dex causes more alpha(2)-selective vasoconstriction in the forearm than clonidine. The similar vasoconstrictor responses to both drugs after prazosin might be explained by the presynaptic effects on norepinephrine release.     

Influence of development and reduction of fat stores on the antilipolytic alpha 2-adrenoceptor in hamster adipocytes: comparison with adenosine and beta-adrenergic lipolytic responses

The response of the hamster adipocyte to various lipolytic (beta-adrenergic) and antilipolytic (alpha(2)-adrenergic and adenosine-dependent) stimuli was studied during the development and after cold-induced regression of fat stores. Alpha(2)-adrenergic binding ([(3)H]clonidine binding sites) was also investigated. Adipocytes came from young animals (4-5 weeks), adults (20-25 weeks), and adults submitted to a 6-week cold exposure (6 degrees C) that promoted a large decrease in fat stores and in fat cell size. The lipolytic response induced by isoproterenol (beta-agonist) was equivalent in the different groups. Adenosine and alpha(2)-adrenergic antilipolytic effects were estimated through the inhibition of theophylline-induced lipolysis by phenylisopropyladenosine and clonidine, respectively. The adenosine effect was unchanged in all the groups. In contrast, the alpha(2)-adrenergic effect, which was not present in young hamsters, increased simultaneously with fat cell size, was fully effective in adult hamsters, and had completely disappeared in small adipocytes from cold-exposed hamsters. In fat cell ghosts, alpha(2)-adrenoceptors ([(3)H]clonidine binding sites), followed similar modifications: they increased with fat cell enlargement and disappeared after cell size reduction following cold exposure. These results suggest that: 1) the increased alpha(2)-adrenergic antilipolytic response which is concomitant with fat cell enlargement could partly explain the growth-related decrease in the previously reported lipolytic effect of epinephrine; 2) the alpha(2)-receptivity of the adipocyte seems to be strictly fat cell size-dependent while the beta-adrenergic and adenosine responses are unaffected; and 3) the regulation in the adipocytes of the adenosine, alpha(2)- and beta-receptors seems to be unrelated.-Carpene, C., M. Berlan, and M. Lafontan. Influence of development and reduction of fat stores on the antilipolytic alpha(2)-adrenoceptor in hamster adipocytes: comparison with adenosine and beta-adrenergic lipolytic responses.     

Distribution of pulmonary vascular pressure as a function of perinatal age in lambs

The distribution of pulmonary vascular resistance (PVR) with respect to compliance was determined using vascular occlusion in isolated lungs from lambs at five ages, from 2 wk before birth to 1 mo of age. The major change in PVR occurred in the pressure gradient across the middle compliant region (delta Pm), which dropped sharply at birth, remained low for 2 wk, and increased at 1 mo. Pulmonary vasoreactivity also varied with ages. Lungs at 0-4 days did not respond to hypoxia and responded poorly to prostaglandin F2 alpha (PGF2 alpha). In contrast, lungs at 13-33 days had significant increases in delta Pm and the gradient across relatively indistensible arterial vessels during hypoxia and increases in all gradients with PGF2 alpha. Ventilation of fetal lungs reduced PVR, mainly because of a 50% reduction in delta Pm. Our results demonstrate that the magnitude and distribution of PVR relative to compliance varied as a function of perinatal age and that pulmonary vasoreactivity depended on postnatal age. The major effect of ventilating fetal lungs was on the middle region.     

Regions of beta 2 and beta 4 responsible for differences between the steady state dose-response relationships of the alpha 3 beta 2 and alpha 3 beta 4 neuronal nicotinic receptors

We constructed chimeras of the rat beta 2 and beta 4 neuronal nicotinic subunits to locate the regions that contribute to differences between the acetylcholine (ACh) dose-response relationships of the alpha 3 beta 2 and alpha 3 beta 4 receptors. Expressed in Xenopus oocytes, the alpha 3 beta 2 receptor displays an EC50 for ACh approximately 20-fold less than the EC50 of the alpha 3 beta 4 receptor. The apparent Hill slope (n(app)) of alpha 3 beta 2 is near one whereas the alpha 3 beta 4 receptor displays an n(app) near two. Substitutions within the first 120 residues convert the EC50 for ACh from one wild-type value to the other. Exchanging just beta 2:104-120 for the corresponding region of beta 4 shifts the EC50 of ACh dose-response relationship in the expected direction but does not completely convert the EC50 of the dose- response relationship from one wild-type value to the other. However, substitutions in the beta 2:104-120 region do account for the relative sensitivity of the alpha 3 beta 2 receptor to cytisine, tetramethylammonium, and ACh. The expression of beta 4-like (strong) cooperativity requires an extensive region of beta 4 (beta 4:1-301). Relatively short beta 2 substitutions (beta 2:104-120) can reduce cooperativity to beta 2-like values. The results suggest that amino acids within the first 120 residues of beta 2 and the corresponding region of beta 4 contribute to an agonist binding site that bridges the alpha and beta subunits in neuronal nicotinic receptors.     

Pharmacological characterization of alpha1- and beta-adrenergic synergism of 5'DII activity in rat brown adipocytes

The role of adrenoceptor subtypes was studied in rat brown adipose tissue (BAT). The type II 5'-deiodinase (5'DII) was activated in response to simultaneous stimulation by beta3- and alpha1-adrenergic agonists, BRL 37344 or CGP 12177, and cirazoline, in brown adipocytes. Inhibition of the alpha1- and beta-adrenergic phenylephrine-stimulated 5'DII activity was obtained by the alpha1-adrenergic antagonists in the order of prazosin >/= wb 4101 > 5-methylurapidil. In comparison, the binding of [3H]prazosin to rat BAT plasma membranes was inhibited by alpha1-adrenergic antagonists in the order of prazosin > WB 4101 = benoxathian > 5-methylurapidil. Although the order of the alpha1-adrenergic competition seemed to be rather typical for the alpha1B-adrenergic receptors, a molecular analysis on adrenoceptor mRNAs should be made to confirm the exact alpha1-adrenergic subtypes at the level of brown adipocytes, since the possibility of a mixture of different receptor subtypes in brown fat cells and/or tissue may interact with the pharmacological characterization. Thus, specific alpha1- and beta-adrenoceptor subtypes participate in the regulation of 5'DII activity in the rat brown adipocytes, and therefore, an impaired alpha1- and beta-adrenergic co-work may be involved in a defective BAT function, e.g., in obese Zucker rats, too. An interesting possibility is that the decreased number of alpha1-adrenoceptors in the BAT of obese Zucker rats is due to the decrease in the alpha1B-adrenoceptor subtype which would further be involved especially in the regulation of BAT 5'DII activity.     

Characterization of a scintillation proximity assay to detect modulators of transforming growth factor alpha (TGF alpha) binding.

A scintillation proximity assay (SPA) for transforming growth factor alpha (TGF alpha) using SPA beads coated with A431 membranes has been studied. Binding of TGF alpha to the beads was characteristic of a receptor interaction. A class of high-affinity receptors for [125I]-TGF alpha (Kd = 0.10-0.26 nM) was detected by competition studies between [125I]TGF alpha and cold TGF alpha and by analysis of association and dissociation rate constants. An antibody to the epidermal growth factor receptor (clone 528) inhibited binding of [125I]TGF alpha (IC50 = 0.20 micrograms/ml), but an anti-TGF alpha antibody (clone 134A-2B3) (less than 25 micrograms/ml) did not block binding. Suramin inhibited [125I]-TGF alpha binding (IC50 = 0.20 mM). The ether lipids 1-O-hexadecyl-2-O-methyl-sn-glycero-3-phosphocholine, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine, and rac-lyso-platelet activating factor inhibited TGF alpha binding (IC50 values of 49, 69, and 57 microM, respectively). SPA is a convenient method for identifying agents that may act by interfering with TGF alpha binding.     

Single channel properties of rat acid-sensitive ion channel-1alpha, -2a,and -3 expressed in Xenopus oocytes

The mammalian nervous system expresses proton-gated ion channels known as acid-sensing ion channels (ASICs). Depending on their location and specialization some neurons express more than one type of ASIC where they may form homo- or heteromeric channels. Macroscopic characteristics of the ASIC currents have been described, but little is known at the single channel level. Here, we have examined the properties of unitary currents of homomeric rat ASIC1alpha, ASIC2a, and ASIC3 expressed in Xenopus oocytes with the patch clamp technique. We describe and characterize properties unique to each of these channels that can be used to distinguish the various types of ASIC channels expressed in mammalian neurons. The amplitudes of the unitary currents in symmetrical Na(+) are similar for the three types of channels (23-18 pS) and are not voltage dependent. However, ASIC1alpha exhibits three subconductance states, ASIC2a exhibits only one, and ASIC3 none. The kinetics of the three types of channels are different: ASIC1alpha and ASIC2a shift between modes of activity, each mode has different open probability and kinetics. In contrast, the kinetics of ASIC3 are uniform throughout the burst of activity. ASIC1alpha, ASIC2a, and ASIC3 are activated by external protons with apparent pH(50) of 5.9, 5.0, and 5.4, respectively. Desensitization in the continual presence of protons is fast and complete in ASIC1alpha and ASIC3 (2.0 and 4.5 s(-1), respectively) but slow and only partial in ASIC2a (0.045 s(-1)). The response to external Ca(2+) also differs: micro M concentrations of extracellular Ca(2+) are necessary for proton gating of ASIC3 (EC(50) = 0.28 micro M), whereas ASIC1alpha and ASIC2a do not require Ca(2+). In addition, Ca(2+) inhibits ASIC1alpha (K(D) = 9.2 +/- 2 mM) by several mechanisms: decrease in the amplitude of unitary currents, shortening of the burst of activity, and decrease in the number of activated channels. Contrary to previous reports, our results indicate that the Ca(2+) permeability of ASIC1alpha is very small.     

Characteristics of the alpha 2/beta-adrenoceptor-coupled adenylate cyclase system and their relationship with adrenergic responsiveness in hamster fat cells from different anatomical sites.

Various studies have shown that the lipolytic response of white adipocytes to catecholamines was dependent on the anatomical origin of these cells. To provide a biological explanation for this phenomenon, we compared hamster white adipocytes, from femoral subcutaneous and epididymal fat, for their lipolytic activities, cAMP responses and adrenoceptor-coupled adenylate cyclase system. Basal and maximal lipolytic responses to the beta-adrenergic (isoproterenol) and the mixed alpha 2/beta-adrenergic (epinephrine) agonists were lower in femoral subcutaneous cells than in epididymal cells, but the alpha 2-adrenergic antilipolytic response to 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline bi-tartate (UK14304) was slightly greater in femoral subcutaneous fat cells than in epididymal fat cells. Identical results were observed for cAMP responses, except for the alpha 2-adrenergic inhibitory response which was identical in both fat deposits. Adrenoceptors studies revealed higher density of inhibitory alpha 2-adrenoceptors 2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline ([3H]RX821002-binding sites) in femoral subcutaneous fat cells than in epididymal fat cells, but identical density of stimulatory beta-adrenoceptors (125I-cyanopindolol-binding sites) and similar subdivision into beta-adrenoceptor subtypes in both adipose deposits. Finally, the level of the alpha-subunits of the stimulatory and inhibitors guanine-nucleotide-binding regulatory proteins, as well as the adenylate cyclase catalytic activity were 40-50% lower in femoral subcutaneous fat cell membranes than in epididymal fat cell membranes. These results suggest that the differences in cAMP and lipolytic responses to catecholamines between epididymal and femoral subcutaneous adipocytes result at least in part from site-related differences in the adenylate cyclase system rather than in the adrenoceptor status.     

Response of the 1-5 day-aged ovine corpus luteum to prostaglandin F2alpha

The hypothesis that, in the ewe, prostaglandin (PG) F2alpha administration on day 3 after ovulation is followed by luteolysis and ovulation was tested using 24 animals. The ewes were treated with a dose of a PGF2alpha analogue (delprostenate, 160 microg) on days 1 (n=8), 3 (n=8) or 5 (n=8) after ovulation, was established by transrectal ultrasonography. Daily scanning and blood sampling were performed to determine ovarian changes and progesterone serum concentrations by radioinmunoassay. The treatment induced a sharp decrease of progesterone concentrations followed by oestrus and ovulation in all ewes treated on days 3 and 5 and in one ewe treated on day 1 (8/8, 8/8, 1/8; P<0.05). Seven ewes treated on day 1 did not respond to PGF2alpha treatment and had an inter-ovulatory cycle of normal length (17.4 +/- 0.5 days). However, the profile of progesterone concentrations during the cycle of these ewes was delayed 1 day (P<0.05) compared with a control cycle. The overall interval between PGF2alpha and oestrus for the 17 responding ewes was 42.4 +/- 2.3 h. In 15 of these ewes the ovulatory follicle was originated from the first follicular wave and the ovulation occurred at 60.8 +/- 1.8 h after PGF2alpha treatment. The other two responding ewes ovulated an ovulatory follicle originated from the second follicular wave between 72 and 96 h after treatment. These results support the hypothesis and suggest that refractoriness to PGF2alpha of the recently formed corpus luteum (CL) may be restricted to the first 1-2 days post-ovulation.