Rashid A. Akhtar

Studies on the properties of triphosphoinositide phosphomonoesterase and phosphodiesterase of rabbit iris smooth muscle

The rabbit iris smooth muscle has been shown to contain triphosphoinositide phosphomonoesterase (phosphatidyl-myo-inositol-4,5-bisphosphate phosphohydrolase, EC 3.1.3.36) and phosphodiesterase (triphosphoinositide inositoltrisphosphohydrolase, EC 3.1.4.11) activities. Under our experimental conditions about 77% of the phosphomonoesterase and 61% of the phosphodiesterase activities were localized in the particulate fraction. The kinetic properties of the enzymes in the microsomal fraction were examined. The enzyme preparation was specific to polyphosphoinositides; it did not attack phosphatidylinositol under the present assay condition. The effects of Ca2+ and Mg2+ were also studied. Although the microsomal enzymes did not require added divalent cations for their activities, both the phosphomonoesterase and phosphodiesterase were appreciably inhibited by 1 mM EDTA. Phosphodiesterase and phosphomonoesterase were stimulated by Ca2+ and Mg2+, respectively. The demonstration of triphosphoinositide phosphodiesterase in the iris muscle, coupled with the findings that this enzyme is activated by Ca2+ and is not influenced by acetylcholine add further support to our previous conclusion (J. Pharmacol. Exp. Ther. (1978) 204, 655--668; J. Neurochem. (1978) 30, 517--525) that an increased Ca2+ influx, following the interaction between the neurotransmitter and its receptor, could act to stimulate the phosphodiesterase, thus leading to increased triphosphoinositide breakdown and increased phosphatidic acid via increased diacylglycerol.

Effects of isoproterenol and forskolin on carbachol- and fluoroaluminate-induced polyphosphoinositide hydrolysis, inositol triphosphate production, and contraction in bovine iris sphincter smooth muscle: Interaction between cAMP and IP3 second messenger systems

We have investigated the effects of isoproterenol (ISO) and forskolin on carbachol(CCh)- and fluoroaluminate (AlF4-)-induced phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, myo-inositol 1,4,5-trisphosphate (IP3) production, 1,2-diacylglycerol, measured as phosphatidic acid (PA) formation, and contraction in the bovine iris sphincter smooth muscle. The data from these studies can be summarized as follows. (1) CCh (20 microM) stimulated significantly PIP2 hydrolysis, IP3 production, PA formation, and contraction. (2) Addition of ISO (0.1-25 microM), which raises the tissue cAMP level, to muscle precontracted with CCh attenuated PIP2 hydrolysis, IP3 production, PA formation and contraction in a time- and dose-dependent manner. (3) AlF4- (10 microM) induced a slow but progressive hydrolysis of PIP2, accompanied by parallel production of IP3, formation of PA, and contraction of the smooth muscle. The effects of AlF4- were dose-dependent and inhibited by deferoxamine, an Al3+ ion chelator. (4) Both forskolin (1-25 microM), which directly stimulates adenylate cyclase, and ISO inhibited the responses induced by AlF4- (10 microM) in a dose-dependent manner. (5) NaF (1-5 mM) had no effect on the activity of phospholipase C (PLC), purified from bovine iris sphincter. Furthermore, phosphorylation of the enzyme by catalytic subunit of protein kinase A had no inhibitory effect on PLC activity against PIP2. In conclusion, neither the muscarinic receptor nor PLC are the target sites for cAMP inhibition; instead the putative G-protein, which couples the activated muscarinic receptor to PLC, may be phosphorylated by cAMP-dependent protein kinase. This could attenuate the stimulation of PLC by the G-protein, thus resulting in inhibition of PIP2 hydrolysis and consequently leading to muscle relaxation. These results demonstrate cross-talk between the cAMP and IP3-Ca2+ second messenger systems and suggest that this could constitute a regulatory mechanism for the process of contraction-relaxation in smooth muscle.

Surgical Sympathetic Denervation Increases α1Adrenoceptor‐Mediated Accumulation of myo‐Inositol Trisphosphate and Muscle Contraction in Rabbit Iris Dilator Smooth Muscle

Abstract: Sympathetic denervation of the iris muscle produces increases in both the breakdown of phosphati‐dylinositol 4,5‐bisphosphate (PIP2) and in muscle contraction in response to norepinephrine (NE). To shed more light on the biochemical basis underlying this supersensitivity we investigated: (1) the effects of NE on PIP, breakdown, measured as myoinositol trisphosphate (IP3) accumulation, and on muscle contraction in normal and denervated rabbit iris dilator; and (2) the effects of denervation on selected biochemical properties of this muscle. The data obtained from these studies can be summarized as follows: (1) The EC50 values (μM) for NE‐induced IP3 accumulation in normal and denervated dilators were 14 and 3, respectively. This accumulation of IP3 was blocked by prazosin (1μM). (2) The EC50 values (μM) for NE‐induced contraction for the normal and denervated muscles were 10 and 0.6, respectively. The NE‐induced muscle contraction was blocked by prazosin (1μM). (3) The t1/2 values (s) for IP3 accumulation in normal and denervated muscles were 31 and 11, respectively, and for contraction the values were 19 and 9, respectively. (4) Denervation increased significantly (15–18%) the basallabelling of phosphoinositides from myo‐[3H]inositol, but not from 32P or [14C]arachidonic acid. (5) Denervation had little effect on the activities of the enzymes involved in phosphoinositide metabolism. However, the activities of protein kinase C and Ca2+‐ATPase increased in the denervated muscle. It is concluded that sympathetic denervation of the iris dilator renders the coupling between α1 receptors and PIP2 breakdown into IP3 and 1,2‐diacylglycerol (DG) more efficient. The NE‐stimulated hydrolysis of PIP2 could then bring about Ca2+ mobilization, necessary for muscle contraction, either directly by causing plasma membrane depolarization or indirectly by IP3 releasing Ca2+ from sarcoplasmic reticulum and by DG activating protein kinase C, or both.

Effects of substance P on inositol triphosphate accumulation, on contractile responses and on arachidonic acid release and prostaglandin biosynthesis in rabbit iris sphincter muscle.

Addition of substance P (10(-7) to 10(-6) M) to rabbit iris sphincter muscle induced: (a) a rapid phosphodiesteratic breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,2-diacylglycerol (DG), measured as phosphatidic acid, and inositol triphosphate (IP3), measured by anion-exchange chromatography; (b) a rapid and strong contractile response, and (c) a rapid release of prostaglandin E2 (PGE2), measured by radioimmunoassay, and rapid release of 14C-labeled arachidonic acid, measured by radiochromatography. These substance P actions are concentration and time-dependent, and are blocked by substance P antagonist, [D-Pro2, D-Trp7,9]SP. The effects of substance P on arachidonic acid release and PG synthesis are not mediated through the cyclo-oxygenase and lipoxygenase pathways. Substance P exerted little effect on PGE2 release by the iris dilator muscle. We conclude that substance P, which is liberated from the sensory nerves that innervate the sphincter region of the iris and plays a role in miosis, may function as a Ca2+-mobilizing agonist in this tissue. Thus, a substance P-induced release of IP3 and formation of DG, a source for arachidonic acid in PG synthesis, followed by Ca2+ mobilization could underlie the mechanism for the biological actions, such as muscle contraction, of the neuropeptide reported in the eye. However, the precise relationship remains to be established.

Species differences in the effects of substance P on inositol trisphosphate accumulation and cyclic AMP formation, and on contraction in isolated iris sphincter of the mammalian eye: differences in receptor density.

The effects of substance P (SP) on inositol trisphosphate (IP3) accumulation, myosin light chain (MLC) phosphorylation, cAMP formation and contraction were studied in iris sphincter smooth muscle of different mammalian species. SP receptor density was also examined in membrane fractions from this tissue. The data obtained can be summarized as follows. (1) In the iris sphincters of rabbit, bovine and pig, SP receptors are coupled to the phospholipase C system, whereas in dog, cat and human these receptors are coupled to the adenylate cyclase system. (2) In those species which employ the phospholipase C system, SP induced IP3 accumulation, MLC phosphorylation and contraction in a dose-dependent manner; in contrast, in those species in which SP induced the formation of cAMP we found the neuropeptide to cause muscle relaxation. The findings on cAMP formation in intact tissue were confirmed in iris sphincter membranes. Both the effect of SP on IP3 accumulation in rabbit and bovine sphincters and its effect on cAMP formation in the dog were blocked by the SP antagonist, (D-Pro2, D-Trp7, 9)-SP. (3) The density of SP receptors in rabbit, bovine and dog were found to be 227, 110.9 and 13.6 fmol mg-1 protein, respectively, and the Kd values were 1.9, 1.8 and 1.3 nM, respectively. (4) Of the neuropeptides investigated SP, neurokinin A and neurokinin B had significant stimulatory effects on IP3 accumulation and on contraction in the rabbit iris sphincter; however, neither neurokinin Y nor the calcitonin gene-related peptide (CGRP) had any effect on these responses. In addition, none of the neuropeptides studied had any effect on IP3 or on contraction in the dog iris sphincter. While it is possible that SP may have dual actions, with the predominant action dependent on the species, the data presented could suggest the presence of two SP receptor subtypes, one coupled to phospholipase C and the other to adenylate cyclase. The results of this investigation indicate major species differences in biochemical and functional responsiveness to SP and in SP receptor density in the iris sphincter of the mammalian eye, and support a modulatory role for the neuropeptide in muscle response in this tissue.

Effects of Corticotropin-(1–24)-Tetracosapeptide on Polyphosphoinositide Metabolism and Protein Phosphorylation in Rabbit Iris Subcellular Fractions

Abstract: Effects of the neuropeptide corticotropin‐(1–24) ‐tetracosapeptide (ACTH) on the endogenous and exogenous phosphorylation of lipids and endogenous phosphorylation of proteins were investigated in microsomes and a 110,000 ×g supernatant fraction [30–50% (NH4)2SO4 precipitate; ASP30–50] obtained from rabbit iris smooth muscle. Subcellular distribution studies revealed that both of these fractions are enriched in diphosphoinositide (DPI) kinase. The 32P labeling of lipids and proteins was measured by incubation of the subcellular fractions with [γ‐32P]ATP. The labeled lipids, which consisted of triphosphoinositide (TPI), DPI, and phosphatidic acid (PA) were isolated by TLC. The microsomal and ASP30–50 fractions were resolved into six and nine labeled phosphoprotein bands, respectively, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The basal labeling of both lipids and proteins was rapid (30–60 s), and it was dependent on the presence of Mg2+ in the incubation medium; in general it was inhibited by high concentrations (>0.2 mM) of Ca2+. ACTH stimulated the labeling of TPI and inhibited that of PA in a dose‐dependent manner, with maximal effect observed at 50–100 μ of the peptide. ACTH appears to increase TPI labeling by stimulating the DPI kinase. Under the same experimental conditions ACTH (100 μM) inhibited significantly the endogenous phosphorylation of six microsomal phosphoproteins (100K, 84K, 65K, 53K, 48K, and 17K). In the ASP30–50 fraction, ACTH inhibited the phosphorylation of three phosphoproteins (53K, 48K, and 17K) and stimulated the labeling of six phosphoprotein bands (117K, 100K, 84K, 65K, 42K, and 35K). The effects of ACTH on lipid and protein phosphorylation are probably Ca2+‐independent; thus the neuropeptide effects were not influenced by either 1 μM EGTA or low concentrations of Ca2+ (50 μ.M). We conclude that a relationship may exist between polyphosphoinositide metabolism and protein phosphorylation in the rabbit iris smooth muscle.

Studies on the mechanism of alteration by propranolol and mepacrine of the metabolism of phosphoinositides and other glycerolipids in the rabbit iris muscle

We have investigated the effects and mechanism of action of propranolol and mepacrine, two drugs with local anesthetic-like properties, on phospholipid metabolism in rabbit iris and iris microsomal and soluble fractions. In the iris, propranolol, like mepacrine [A. A. Abdel-Latif and J. P. Smith, Biochim, biophys. Acta 711, 478 (1982)], stimulated the incorporation of [14C]arachidonic acid ( [14C]AA) into phosphatidic acid (PA), CDP-diacylglycerol (CDP-DG), phosphatidylinositol (PI), the polyphosphoinositides (poly PI) and DG, and it inhibited that of phosphatidylcholine (PC), phosphatidylethanolamine (PE), triacylglycerol (TG) and the prostaglandins. Similarly, mepacrine, like propranolol [A. A. Abdel-Latif and J. P. Smith, Biochem. Pharmac. 25, 1697 (1976)], altered the incorporation of [14C]oleic acid, [3H]glycerol, 32Pi and [14C]choline into glycerolipids of the iris. Time-course studies in iris muscle prelabeled with [14C]AA showed an initial decrease in the production of DG and a corresponding increase in that of PA by the drugs, followed by an increase in accumulation of DG at longer time intervals (60-90 min). The above findings are in accord with the hypothesis that these drugs redirect glycerolipid synthesis by inhibiting PA phosphohydrolase. Propranolol and mepacrine stimulated the activities of DG kinase and phosphoinositide kinases and inhibited that of DG cholinephosphotransferase. The drugs had little effect on the activity of DG acyltransferase. It is concluded that propranolol and mepacrine redirect glycerolipid metabolism in the iris by exerting multiple effects on the enzymes involved in phospholipid biosynthesis. We suggest that these drugs could exert their local anesthetic-like effects by effecting an increase in the synthesis of the acidic phospholipids (PA, PI and the poly PI) and subsequently the binding of Ca2+- to the cell plasma membrane.

[3H]Quinuclidinyl benzilate binding to muscarinic receptors and [3H]WB-4101 binding to alpha-adrenergic receptors in rabbit iris: comparison of results in slices and microsomal fractions

The binding characteristics of [3H]-(l)-quinuclidinyl benzilate (QNB) and [3H]WB-4101 to microsomal fractions and slices from rabbit iris muscle were compared. [3H] QNB binding to both microsomal fractions and muscle slices was of high affinity and low capacity and was displaced by muscarinic ligands. The equilibrium dissociation constants (KD) for [3H]QNB binding to microsomes and slices were 0.069 nM and 1.97nM, respectively. This shift to a higher value for the Kp of the microsomal fraction compared with that of the slices was also observed lor the association rate constants (KI) and inhibition constants (KJ), but not for the dissociation rate constants (K−1). Kinetic studies on the binding characteristics of [3H]WB-4101 revealed high affinity sites with KD values of 2.33 and 10.19 nM for microsomal fractions and slices, respectively. The findings of comparable binding patterns for [3H]QNB and [3H]WB-4101 binding to microsomal fractions and intact muscle slices argue against the possibility of alterations in receptor properties following tissue disruption. It is proposed that the differences in receptor-mediated biochemical responses that are seen between intact tissue and cell-free homogenates, such as the ‘phosphoinositide effect’, are more likely to be due to alterations in receptor function, e.g. changes in ionic permeabilities, rather than to actual changes in receptor properties.

Effects of norepinephrine and 5-hydroxytryptamine on phosphoinositide-PO4 turnover in rabbit cornea.

We have investigated: (a) Phospholipid composition and phosphoinositide-PO4 turnover in rabbit cornea tissues; and (b) the effects of adrenergic and serotonergic agonists on breakdown of phosphoinositides in the rabbit cornea. The data obtained from these studies can be summarized as follows: (1) in the cornea phosphatidylcholine and phosphatidylethanolamine constitute about 55%, phosphatidylinositol (PI) 10%, and phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) comprise about 1% each of the total phospholipids; (2) incubation of cornea in 32Pi-containing medium resulted in incorporation of radioactivity in tissue phospholipids. The radioactivity was highest in PIP2 (39%), followed by PI (19%), PIP (16%) and PA (5% of the total radioactivity). When compared with stroma and endothelium, the cornea epithelium was most active in phosphoinositide metabolism; (3) addition of norepinephrine (NE) or 5-hydroxytryptamine (5-HT), 200 microM each, to 32P-labeled cornea resulted in a loss of radioactivity in PIP and PIP2 by about 12- and 20%, respectively. Concomitantly, the radioactivity in PA and PI was increased by 44- and 66%, respectively. The effects of the neurotransmitters were time- and concentration-dependent. When added to the cornea labeled with myo [3H] inositol, NE and 5-HT increased the production of labeled myo-inositol phosphates; (4) prazosin (20 microM), but not yohimbine or propranolol, blocked the effects of NE. Similarly, the effects of 5-HT were antagonized by methysergide (20 microM) and ketanserin (10 microM) but not by prazosin. These data demonstrate that NE and 5-HT stimulate phospholipase C-mediated hydrolysis of PIP2 into diacylglycerol (DG) and myo-inositol trisphosphate (IP3). Furthermore, the effects of NE and 5-HT are mediated by alpha 1-adrenergic and 5-HT2 receptors, respectively. It is suggested that IP3, by releasing Ca2+ from ER, and DG, by activating protein kinase C, may function as second-messenger molecules which may participate in agonist-induced functional responses, including chloride transport, in the cornea epithelium.

Polyphosphoinositides and Muscarinic Cholinergic and α1-Adrenergic Receptors in the Iris Smooth Muscle

An attempt was made in this brief review first to recount some of our early studies which culminated in the characterization of the polyphosphoinositide (phosphatidylinositol 4-monophosphate, PIP and phosphatidylinositol 4,5-bisphosphate, PIP2) effect in the iris smooth muscle, and second to present more recent data on the rapid breakdown of 32 P-prelabeled polyphosphoinositide (PPI) and release of [3H]myo-inositol phosphates by carbachol (CCh) in this tissue. The PPI effect is defined as the agonist-stimulated breakdown of PIP2 into diacylglycerol, measured as labeled phosphatidate, and inositol trisphosphate (IP3). These early findings included: (1) the demonstration of an agonist-stimulated breakdown of PIP2 which occurred at relatively short time intervals (2.5–10 min), when compared to the phosphatidylinositol (PI) effect reported in a variety of tissues; (2) the demonstration of the PPI effect in vivo, in response to electrical stimulation of the sympathetic nerve of the eye; (3) the demonstration, through pharmacologic and adrenergic denervation supersensitivity studies that PIP2 breakdown is linked to muscarinic cholinergic and aladrenergic receptors; (4) the demonstration that phosphodiesteratic cleavage of PIP2 into diacylglycerol and I P3, by PIP2 phosphodiesterase, is the molecular mechanism underlying the PPI effect; (5) the demonstration of some requirement for \(C{{a}^{{{{2}^{ + }}}}}\), derived mainly from studies on the inhibitory effects of EGTA and \(C{{a}^{{{{2}^{ + }}}}}\) ionophore A23187 on this phenomenon; however, the recent finding that the cationophore-stimulated breakdown of PIP2 is blocked by prazosin leads us now to conclude that while the PPI effect in the iris needs some \(C{{a}^{{{{2}^{ + }}}}}\), it is not regulated by intracellular \(C{{a}^{{{{2}^{ + }}}}}\); (6) the demonstration of a close correlation between agonist-stimulated PIP2 breakdown and agonist-induced muscle contraction, which led us to suggest that the agonist-stimulated PIP2 breakdown is an early event in the pathway which leads from receptor activation to muscle response. Data are also presented which demonstrate that in the iris, the breakdown of labeled PIP2 and release of IP3 by CCh occur within 15 s; in contrast the release of IP occurred at longer time intervals (>1 min). Thus after incubation for 15 s with CCh there was 48% loss of 32P radioactivity from PIP2 in 32P-labeled iris, 81% increase in IP3 release and no change in the release of IP in iris prelabeled with [3H]inositol. These data suggest that agonist-stimulated PIP2 breakdown is probably involved in the mechanism of both the phasic (fast) and tonic (slow) components of the contractile response. Neither 2-deoxyglucose nor Li+, when added for short time intervals (10 min), had any influence on the PPI effect. In accord with our previous studies we conclude that the phosphodiesteratic cleavage of PIP2 is an early (initial) event in the pathway which leads from activation of \(C{{a}^{{{{2}^{ + }}}}}\)-mobilizing receptors to muscle response.