Microsomal (Na- +K+)-activated ATPase from frog skin epithelium. Cation activations and some effects of inhibitors.

A method is described for the extraction of microsomal ouabain-sensitive (a- + K+)-activated ATPase from separated frog skin epithelium. The method yields a microsomal fraction containing (Na+ K+)-stimulated activity in the range of 30- 40 nmol - mg -1 - min -1 at 26 degrees C. This portion which is also ouabain sensitive, is about half of the total activity in media containing Mg2+, Na+ and K+. These preparations also contain Mg2+-dependent or Ca2+-dependent activities which are not additive and which are not significantly affected by ouabain, Na+, K+ or Li+. The activations of the ouabain-sensitive ATPase activity by Mg2+, Na+, and K+ are similar to those described in other tissues. It is found that Li+ does not substitute for Na+ as an activator but in high concentrations does produce partial activation in the presence of Na+ with no K+. These results are pertinent to the reported observations of ouabain-sensitive Li+ flux across frog skin. It is concluded that this flux is not apparently due to a direct activating effect of Li+ on the sodium pump.

Drug responses of adenylate cyclase in iris-ciliary body determined by adenine labelling.

The intracellular adenine nucleotide pool of rabbit iris-ciliary body was labelled by uptake of 3H-adenine in vitro. A variety of agents were tested for their ability to stimulate or inhibit the incorporation of radioactivity into cyclic AMP formed from ATP labelled with 3H-adenine. Isoproterenol, vasoactive intestinal peptide, forskolin, and prostaglandin E2 stimulated incorporation of label 3-10-fold in 15-20 min compared with paired tissues not treated with hormone, whereas histamine, serotonin, substance P, and bradykinin were inactive. Clonidine, alpha-methylnorepinephrine, and dopamine decreased the rate of incorporation of label into the cyclic-AMP pool in tissues that showed high spontaneous basal rates. In low-basal tissues these drugs were inactive by themselves but clonidine and alpha-methylnorepinephrine blocked the stimulation effected by isoproterenol. The findings indicate that several receptor-coupled adenylate cyclase systems are present in ICB and that dual adrenergic control of adenylate cyclase through positive and negative coupling of adrenergic receptors probably occurs. The negatively coupled adrenergic receptors appear to be similar to the alpha 2-subclass of adrenergic receptor described in other tissues. These observations suggest a role for the large number of alpha 2-adrenergic-binding sites found in albino rabbit iris-ciliary body by ligand binding assays.

Calmodulin activated adenylyl cyclase in ciliary processes: additivity of calcium and cyclic adenosine monophosphate signals on intraocular pressure response of the rabbit eye.

PURPOSE: The regulation of adenylyl cyclase by multiple signal systems was investigated by biochemical studies of the enzyme in ciliary processes and by in vivo intraocular pressure responses in the rabbit eye. METHODS: Adenylyl cyclase enzyme activity was determined by radiometric assay using alpha-32 P-ATP as substrate. Drugs were administered to the rabbit eye by intravitreal injection or topical application, and intraocular pressure was measured by pneumatonography. RESULTS: Adenylyl cyclase activity in the membrane/particulate fraction of the rabbit ciliary process was activated by calmodulin in the presence of Ca2+, Co2+, or Mn2+, and inhibited by the calmodulin antagonist calmidazolium. The activity was additive to stimulations of adenylyl cyclase by the activating G-protein (Gs) via isoproterenol or vasoactive intestinal peptide receptors, and by forskolin. The biochemical findings were supported by in vivo correlation experiments with intravitreal injection of MnCl2, and by topical treatment with the Ca(2+)-mobilizing alpha 1-adrenergic agonist phenylephrine and with agents affecting cyclic adenosine monophosphate levels (forskolin, isobutylmethylxanthine). The intraocular pressure response was augmented by combining threshold doses of phenylephrine with threshold doses of either forskolin or isobutylmethylxanthine. The maximal and most prolonged response (a decrease in intraocular pressure of 4 mm Hg for up to 8 hr) was obtained by combination treatment with all three agents at doses that produced no significant effect by themselves. CONCLUSIONS: These findings provide a biochemical mechanism for ocular hypotensive drugs having both a Ca(2+)-mobilizing activity as well as increasing cyclic adenosine monophosphate levels by receptor-coupled stimulation of adenylyl cyclase.

Effects of inorganic ions on rabbit ciliary process adenylate cyclase.

The interaction of several inorganic ions (Mn2+, Ca2+, VO3-, and F-) with adenylate cyclase in the albino rabbit ciliary process particulate fraction was studied. Effects of these substances were determined on three activity states of the enzyme (basal, Gs-protein stimulated activity via isoproterenol activation of beta-adrenergic receptors, forskolin-activated catalytic unit) in the presence of 3 mM Mg2+, 1 mM EDTA and 0.2 mM EGTA. A different pattern of effects was found for each of the four ions. Addition of Mn2+ (2 mM) increased all three responses; basal by up to ten-fold, isoproterenol and forskolin by four to five-fold. Added Ca2+ (1 mM) increased basal by two to three-fold, but inhibited isoproterenol and forskolin responses by 25-50%. Added vanadate (1-10 mM) increased basal by two to four-fold, had no effect on the isoproterenol response, but doubled the forskolin response at 3 mM. Added F- (10 mM) increased basal by 30-40-fold, decreased the isoproterenol response and potentiated the forskolin response. The response to F- which directly activates G-proteins was much greater than that of non-hydrolysable GTP analogs, which also directly activate G-proteins. The results suggest that more than one type of adenylate cyclase and/or several modes of regulation of adenylate cyclase with different ionic requirements may be present in ciliary process membranes.

Ocular hypotension in the rabbit. Receptor mechanisms of pirbuterol and nylidrin.

Pirbuterol and nylidrin, both purported sympathomimetic amines, reduced intraocular pressure (IOP) when given topically (50 microliter, 0.1%) to albino rabbits. Pirbuterol increased the cyclic-AMP concentration in aqueous humor by a factor of 3.25, while nylidrin had no effect on aqueous cyclic-AMP nor on adenylate cyclase activity of iris-ciliary body membranes assayed in vitro. Studies of the receptor affinity of pirbuterol, timolol and nylidrin were carried out on iris-ciliary body membranes by competition binding with radioactive ligands. Four ligands were used that appear to label separate subpopulations of adrenergic receptors; dihydroalprenolol (beta-receptors), WB-4101 (alpha 1-receptors) prazosin (alpha 1-receptor subpopulation) and yohimbine (alpha 2-receptors). Pirbuterol and timolol showed exclusive selectivity for beta-receptors with high affinities (Kd 12.6 and 0.48 nM, respectively) compared with other adrenergic receptor populations in iris-ciliary body. Nylidrin had high affinities for beta-receptors (Kd 22 nM) and for the subpopulation of alpha 1-receptors labelled by prazosin (Kd 6.5 nM), but showed 100-fold lower affinity and complex binding characteristics to the two other classes of alpha-adrenergic receptor sites labelled by WB-4101 and yohimbine, respectively. The results show that pirbuterol and timolol are highly beta-receptor selective and that hypotensive responses to these drugs are not mediated by the other classes of alpha-adrenergic receptor determined in this study. However, the hypotensive response to nylidrin may be related to its prazosin-like (alpha 1-receptor) antagonist properties with additional activity at beta-receptors.

Effects of Al3+ and Be2+ ions combined with NaF on ciliary process adenylyl cyclase activity and aqueous humor dynamics in the rabbit eye.

PURPOSE: The activity of Al3+, Ga3+, and Be2+ ions in the presence of NaF to directly activate G-proteins was investigated by their potentiative effect on forskolin (FSK)-activated adenylyl cyclase in rabbit ciliary process membranes and their effects on aqueous humor dynamics in vivo. METHODS: Adenylyl cyclase (AC) was determined by radiometric conversion of ATP to cAMP by the particulate fraction of rabbit ciliary processes. Intravitreal injections of sterile solutions of analytical grade salts were made into the center of the vitreous in a volume of 20 microliters. Intraocular pressure, aqueous humor flow, and uveoscleral outflow measurements were made by pneumatonometry, fluorophotometry, and fluorescein-dextran method, respectively. Outflow facility was determined by tonography in the intact eyes and by two-level constant pressure perfusion in cannulated eyes. RESULTS: Both Al3+ (EC50, 40 mumol/l) and Be2+ (EC50, 11 mumol/l) in the presence of 0.5-2 mM NaF activated the stimulatory G-protein Gs. Ga3+ was ineffective and did not antagonize the activation by Al3+. Intravitreal injections of Al3+ (1 mumol/eye) or Be2+ (0.5 or 1 mumol/eye) had no significant intraocular pressure (IOP) effect, nor did 1.5 or 3 mumol/eye of NaF, but when either cation was injected together with NaF, IOP decreased by up to 40% for up to 140 hr. At the time of maximum IOP effect (72 hr) aqueous humor flow determined by fluorophotometry was decreased in BeCl2+ NaF-treated eyes by 40% relative to BeCl2-treated eyes; however, tonographic facility of outflow was unaffected. Uveoscleral flow was also decreased by 38% in BeCl2+ NaF treated eyes. CONCLUSIONS: These findings support the hypothesis that Gs activation of ciliary process adenylyl cyclase decreases aqueous humor formation rate in rabbit eyes, and that activation of G-proteins mediates contraction of ciliary muscles causing a decrease of aqueous humor outflow via the uveoscleral route. The results suggest that G-proteins putatively involved in trabecular facility changes are less sensitive to activation by BeF3- than are other parameters of aqueous humor dynamics.

Effects of isoxazolium cations on some isolated muscle preparations.

1. The quaternary ammonium compound, N,t-butyl-5-methyl isoxazolium perchlorate (BIP), an anionic group reagent, initially causes contractions of the rat phrenic-nerve diaphragm, guinea-pig ileum and rabbit aortic strip preparations in vitro.2. In addition, the drug produces an irreversible block of indirectly elicited twitch responses in the diaphragm and of contractions induced by acetylcholine, methylfurmethide, dimethyl-phenylpiperazinium, 5-hydroxytryptamine (5-HT), histamine, angiotensin and pilocarpine in the ileum, while direct electrical stimulation of the diaphragm and contractions of the ileum to Ba and K ions are relatively unaffected.3. BIP is also an irreversible inhibitor of acetylcholinesterase but not of butyrylcholinesterase.4. On rabbit aortic strip preparations, responses to histamine, noradrenaline and 5-HT were differentially sensitive to irreversible blockade by BIP.5. Diphenhydramine, used in conditions which gave complete protection of the histamine response to irreversible block by dibenamine, did not protect against the blocking action of BIP but increased the blockade.6. These results suggest that BIP reacts covalently with anionic groups which mediate receptor initiated stimuli. The isoxazolium group may be useful in conferring irreversible properties by its substitution in drug molecules for the pyrrole or pyrrolidine group.

Endotoxins in cholera and pertussis toxins interfere with in vivo responses to these agents in the albino rabbit eye.

Intravitreal injections of cholera or pertussis toxin (CTX or PTX, 0.5-1 microgram/eye) decreased intraocular pressure (IOP) up to 50% in the albino rabbit eye, which lasted up to six days. Both toxins were active on G-proteins as determined by in vitro and in vivo effects on ciliary process adenylyl cyclase activity and by ADP ribosylation of G-protein alpha-subunits with 32P-NAD. However, forty-two hours after toxin injection aqueous humor proteins increased from control levels of 0.8-1.2 mg/ml to 8-25 mg/ml. Both toxins contained 1-3 parts per thousand endotoxin sufficient to cause the IOP and aqueous humor protein responses observed. We conclude that the in vivo responses to intraocular CTX or PTX obtained from commercial sources may not provide unequivocal evidence for the role(s) of G-proteins in aqueous humor dynamics, and must be interpreted with caution.

Atrial natriuretic peptide (ANP), guanylate cyclase, and intraocular pressure in the rabbit eye.

Synthetic rat atrial natriuretic peptide (ANP) was examined for effects on guanylate-and on adenylate cyclase activity in ciliary process homogenates and for effects on intraocular pressure in the albino rabbit eye. Ciliary process guanylate cyclase was associated predominantly with the particulate fraction and was partially activated by ANP (EC50, approximately 1 nM) relative to a maximal dose of Na Nitroprusside (2 uM), whereas particulate adenylate cyclase (basal as well as forskolin-stimulated activity) showed no responses to ANP at doses up to 0.3 uM. Particulate cAMP phosphodiesterase activity was stimulated by low doses of cGMP (1-5 uM) in ciliary processes. Thus, ANP, acting via guanylate cyclase, has the potential to regulate phosphodiesterase activity and indirectly decrease cAMP levels in membranes derived from ciliary processes. Intravitreous injection of ANP (2-4 ug/eye) caused a small decrease (1-1.5 mm Hg) in intraocular pressure measured 16-24 hours after injection but the pressure had returned to normal by 40 hours. The findings demonstrate regulation of biochemical and pharmacological responses by ANP in the albino rabbit eye suggesting that this peptide may play a physiological role in secretory functions of ciliary processes.

Role of G-proteins in ciliary process adenylyl cyclase responses of the albino rabbit eye.

After intravitreal injections of cholera or pertussis toxin (CTX or PTX, 0.5 -1 microgram/eye) into the albino rabbit eye, the in vitro responses of ciliary process adenylyl cyclase (AC) to isoproterenol, vasoactive intestinal peptide (VIP), and forskolin (FSK) were increased 21-40% for PTX, but for CTX-injected eyes AC responses to fluoroaluminate, VIP and FSK decreased 70-50%. The increased responses after PTX suggests that this toxin blocked an inhibitory Gi control of AC that is present in the control tissue. However, prolonged (> 24 hr) in vivo exposure to CTX appears to down-regulate the AC enzyme. In contrast to the in vivo findings, AC responsiveness was unaffected by PTX pre-treatment of membranes in vitro, while CTX pre-treatment increased basal activity (+600%), and the FSK response (+30%), but decreased responsiveness to fluoroaluminate, VIP and isoproterenol by 88-56%. Treatment of ciliary process membranes with 32P-NAD and CTX or PTX followed by SDS-PAGE autoradiography of labelled proteins gave two bands for the G-protein alpha-subunits of Gs (45, 56 kDa) and one broad band centered at 40 kDa for Gi-type subunits respectively. Western blots using specific antibodies showed the presence of Gi type I or III, but no detectable Gi type II or Go in rabbit ciliary processes. We conclude that the changes in adenylyl cyclase enzyme responses after intraocular CTX or PTX may not correlate with cAMP levels and intraocular pressure effects. However, the in vitro biochemical data on AC responses and on G-proteins provide evidence for dual regulation of ciliary process AC by activating and inhibitory G-proteins.

Adrenergic receptor subtypes in rabbit iris-ciliary body membranes: classification by radioligand studies.

Receptor subclasses in iris-ciliary body cell membranes were determined by direct binding of radioactive dihydroalprenolol (DHA), yohimbine (YOH), WB-4101 WB) and prazosin (PRZ), classified respectively as beta 1 + beta 2, alpha 2, alpha 1, and alpha 1 subtype selective ligands (based on binding to brain adrenergic receptors). Specific binding was defined with appropriate unlabelled agonist and antagonist drugs in each case. Binding data were analysed by library programs of the PROPHET computer system. Subclass specificity was also determined indirectly by binding competition of the labelled ligand (at a three- to 10-fold Kd concentration) with increasing concentrations of 'cold' agonist or antagonist. Ligand binding parameters of unlabelled drugs were obtained from Dixon and Scatchard plots of the data. The adrenergic receptor density of iris-ciliary body is approx. 600 fmol mg-1 protein, of which 20-25% are primarily beta 2 receptors. Three distinct subpopulations of alpha-receptors, representing 10, 25 and 40-45% of the total, bind PRZ, WB and YOH, respectively, each with high specificity for its corresponding ligand but with 10- to 1000-fold lower specificity for the other two ligands. The majority of alpha-adrenergic receptors are of the alpha 2-subtype. A small population of receptors are similar to vascular postsynaptic alpha 1-receptors, while a larger subpopulation may have characteristics intermediate between alpha 1- and alpha 2-subtypes.

Interactions between forskolin, GS, and divalent cations on ciliary process adenylyl cyclase and intraocular pressure in the rabbit eye.

The major adenylyl cyclase activity (AC) of rabbit ocular ciliary processes was investigated by dose-response analysis with respect to interactions of its regulators [the stimulatory G-protein (alpha S) and forskolin] under both saturating and suboptimal divalent cation (M2+) conditions. alpha S was generated directly with fluoroaluminate or via receptors for vasoactive intestinal peptide or isoproterenol. Forskolin and alpha S when liganded simultaneously to AC at suboptimal M2+, potentiated maximal enzyme activity (approximately twice the sum of the separate activities), while the responses were additive (no potentiation) under saturating M2+ conditions. The results demonstrate that the three ligands (forskolin, M2+ and alpha S) mutually interact in a cooperative manner as follows: (1) The apparent k(act) of forskolin for AC was decreased by 1.7-1.9 log units when alpha S was liganded to AC. (2) Conversely, with forskolin liganded to AC the apparent k(act) of alpha S for AC also decreased by a factor of at least 3-4. (3) The nature of the divalent cation M2+ had no effect on the k(act) of alpha S for AC alone and for the AC:FSK complex, and also did not affect the apparent k(act) of forskolin for AC alone, or for the AC: alpha S complex. The nature of M2+ (Mg2+ or Mn2+) affected only the maximal catalytic rate (Emax). (4) Potentiation of adenylyl cyclase by alpha S + FSK was demonstrable in vivo by the increased ocular pressure response of the rabbit eye when a low topical dose of isoproterenol (125 ng per eye) and a subthreshold dose of forskolin (100 micrograms per eye) were given in combination.

Manganous chloride stimulation of adenylate cyclase responsiveness in ocular ciliary process membranes.

Manganous chloride was compared with magnesium chloride in supporting maximal stimulations of the adenylate cyclase system in ocular ciliary process membranes by isoproterenol, vasoactive intestinal peptide, sodium fluoride, guanosine 5'-(beta, gamma-imino) triphosphate (GppNHp), or forskolin, and in supporting synergism between isoproterenol and forskolin. The primary effect of Mn2+ (2 mM) was due to an interaction at the catalytic unit. Mn2+ had no significant effect on the function of the GTP-binding stimulatory G-protein (Gs) which couples beta-adrenergic receptors to the catalytic unit of adenylate cyclase. However, Gs-protein function was impaired by Mn2+ relative to Mg2+ when GppNHp was used instead of GTP as the ligand for the Gs-protein. Compared with Mg2+, Mn2+ caused a 4-5.5-fold increase in adenylate cyclase responsiveness to all the activators tested (except GppNHp, where the increase was 2.5-3.5-fold). Thus Mn2+ ions appeared to be intrinsically more effective at divalent cation binding sites on the catalytic unit that control its enzymatic activity. Ciliary process membranes differ from erythrocyte and S49 lymphoma cell membranes where 2 mM Mn2+ strongly inhibits hormone-Gs-protein-mediated stimulations of adenylate cyclase. Divalent cations bound to the catalytic unit also affected the degree of synergism between hormone-activated Gs and forskolin to stimulate adenylate cyclase activity. In the presence of MgCl2 all effective doses of isoproterenol and forskolin in combination showed marked synergism. In contrast, with MnCl2 there was no synergism with high-dose isoproterenol-forskolin combinations, which gave only additive responses.

Vasoactive intestinal peptide and intraocular pressure: adenylate cyclase activation and binding sites for vasoactive intestinal peptide in membranes of ocular ciliary processes.

Vasoactive intestinal peptide (VIP)-responsive adenylate cyclase and VIP binding sites were investigated in membranes prepared from ciliary processes dissected from albino rabbit eyes. High-affinity binding sites for VIP (Kd, 0.95 nM; 607 fmol/mg of protein), in addition to beta adrenergic sites labeled by dihydroalprenolol (Kd, 0.48 nM; 123 fmol/mg of protein), were present. Activation of adenylate cyclase by VIP had a Ka of 65 nM, and the maximal response was 3.3-fold greater than that for I-isoproterenol (Ka, 102 nM). A peptide fragment of VIP (sequence 10-28) was inactive in all assays and did not inhibit VIP-stimulated adenylate cyclase at 10 microM. Responses to VIP and isoproterenol in combination were additive at lower doses but less than additive at maximal doses. Responses to VIP in combination with a low dose of forskolin (0.1 microM) were potentiated at all dose levels, whether assays were done in presence of MgCl2 or MnCl2. VIP- and forskolin-activated adenylate cyclase was associated with the nonpigmented epithelial cell fraction and not with pigmented epithelial cells separated on Percoll density gradients after dissociation of cells from processes by collagenase digestion. Intravitreous injection of 10 nmol of VIP into the rabbit eye caused a maximal reduction in intraocular pressure at 40 to 50 hr lasting beyond 72 hr. VIP-responsive and beta adrenergic-responsive adenylate cyclase are present on the same cell type (nonpigmented epithelial cells) and appear to share components of the adenylate cyclase system in the same membrane. VIP may participate in the physiologic regulation of aqueous humor secretion at the level of the epithelial cell membrane.

Detection of anti-acetylcholine receptor factors in serum and thymus from patients with myasthenia gravis.

Since the blood and thymus of patients with myasthenia gravis may contain inhibitors of neuromuscular transmission that affect acetylcholine receptors of striated muscle, we used denervated rat muscle to test for inhibitors in 43 serums and 18 thymus glands from such patients. Seven per cent of serums inhibited the binding of 125l alpha-bungarotoxin to triton-solubilized receptors; 65 per cent interfered with binding of toxin-labeled receptors to concanavalin-A coupled to Sepharose gel, and 85 per cent formed IgG-receptor complexes detectable by immunoprecipitation. Serum inhibitory activity varied widely among patients with similar clinical manifestations and was not correlated with duration of myasthenia gravis or thymectomy. Among thymus extracts, 44 per cent were inhibitory in the concanavalin-A binding assay, whereas 72 per cent contained anti-receptor IgG. Thus, serums from patients with myasthenia gravis contain more than one anti-receptor factor.

Alpha-adrenergic antagonists: correlation of the effect on intraocular pressure and on alpha 2-adrenergic receptor binding specificity in the rabbit eye.

Six alpha-adrenergic antagonists, which have a range of selectivities for alpha 1- and alpha 2-adrenoreceptor subtypes, were compared with respect to their ability to reduce intraocular pressure (IOP) after topical application to the rabbit eye, and their affinity and selectivity for alpha 2-adrenoreceptors, as determined by binding to membranes prepared from rabbit iris-ciliary body. A routine assay for alpha 2-adrenoreceptors using [3H]-rauwolscine was developed for this purpose. ICB contained 200-300 fmol (mg protein)-1 alpha 2-adrenoreceptors which represents approximately two-thirds of the total number of alpha-adrenoreceptor sites present in this tissue. All six antagonists bound at alpha 2-adrenergic receptor sites in an apparently simple competitive manner. The Kd for three of the drugs was about 10 nM (rauwolscine, yohimbine, WB-4101) and the Kd for the other three was greater than 3500 nM (prazosin, corynanthine, thymoxamine). However, all six antagonists were effective ocular hypotensive agents when given topically in a 50 microliter dose of 1% (w/v) concentration. The ability of alpha-adrenergic antagonists to lower IOP in the rabbit did not correlate with a single alpha-receptor subtype and appears to involve at least two separate mechanisms, one mediated by alpha 2-adrenergic receptors and one mediated by alpha 1-adrenergic receptors.

Polylysine stimulated protein phosphorylation in rabbit ciliary processes: casein kinase activities.

Protein phosphorylation in response to polylysine was investigated in vitro in rabbit ciliary process homogenates by SDS-PAGE autoradiography. The degree of phosphorylation was greater in the soluble/cytoplasmic fraction than in the particulate fraction and was antagonized by heparin. Time and dose-dependent studies indicated several different kinetic patterns of phosphorylation/dephosphorylation among the approximately 15 significantly 32P-labeled bands found in each fraction. These results are consistent with phosphorylation of endogenous substrates by casein kinase II, and dephosphorylations by type I and type II phosphoprotein phosphatase enzymes. The presence of EGF receptors in ciliary processes was indicated by high affinity (kD < 0.5 nM) binding sites and by intraocular pressure and blood-aqueous barrier responses to injection of low doses of EGF (100 ng per eye). EGF did not stimulate protein phosphorylation in ciliary process homogenates in vitro. The results show that casein kinase II is a significant kinase activity in ciliary processes and may have a modulatory role on signal transduction proteins involved in cellular response to hormones.