Pharmacological Studies of Histamine Receptors in the Anterior Uvea

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Kulkarni, Kaustubh Hridaynath
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Although histamine has been localized in ocular tissues, its role in aqueous humor dynamics and the regulation of intraocular pressure (IOP) is yet to be fully determined. The overall objective of the present study was two-fold: (a) to examine the prejunctional action of exogenous histamine on sympathetic neurotransmission in the bovine iris in uiho and (b) to investigate the postjunctional effect of histamine on the contractility of the isolated bovine iris. For prejunctional studies, isolated bovine and human hemi-irides were prepared for studies of electrically-evoked PH]-norepinephrine (PH]NE) release using the superfusion method. In postjunctional studies, paired iris muscle strips were set up in organ baths for measurement of isometric contractile/relaxant tension generated in response to drugs. Electrical field stimulation of irides preloaded with PH]-norepinephrine (PH]NE) caused an increase in tritium efflux over baseline. Elistamine and receptor selective agonists caused concentration-dependent inhibition of electrically-evoked PEfJNE release with the following rank order of potency: imetit > R-a-methylhistamine > histamine. In human iris-ciliary bodies, equipotent concentrations of R-a-methylhistamine (0.3 and 1 pM) also caused an inhibition of piT]NE that was of similar magnitude to those observed in bovine tissues. To further confirm the pharmacological subtype of prejunctional histamine receptors regulating pEl]NE release, effects of EE- and/or EE-receptor antagonists were examined on the histamine response. Both clobenpropit and thioperamide completely blocked the inhibition of PH]NE release caused by the R-a-methylhistamine and imetit, respectively. These results indicate that the prejunctional receptor that mediates inhibitory effects on sympathetic neurotransmission belongs to the Ha-receptor subclass. To determine if both prejunctional Ha-receptors and a2-adrenoceptors coexist on the same nerve terminal, I examined the combined effect of R-a- methylhistamine and clonidine on field-stimulated PHjNE release. Effects caused by maximally effective concentrations of clonidine were not additive with those elicited by corresponding concentrations of R-a-methylhistamine suggesting that prejunctional Ha- and a2-adrenoceptors coexist at neurotransmitter release sites and possibly share a common mechanism for inhibition of NE release. In postjunctional studies, I examined the both contractile and relaxant effect of histamine on isolated bovine iris smooth muscle. Histamine (10 nM - 10 pM) caused a concentration-related contractile response in the isolated bovine iris with an EC50 value of 557 nM. Inhibition of phospholipase A2 with quinacrine and cyclooxygenase (COX) with indomethacin caused a concentration-dependent attenuation of contractile responses to histamine. Taken together, these observations indicate that histamine induced contractions of isolated bovine irides involves activation of the pathway leading to the synthesis of prostanoids. To investigate the pharmacological profile of the histamine receptor that mediates contractile responses to this autacoid, I compared the contractile effect of histamine with those of selective histamine Hi- receptor agonist HTMT, hh-receptor agonist R-a-methylhistamine and the H3- /Hi-receptor agonist, imetit. All agonists tested caused concentration-dependent contractile responses with the following rank order of potency: histamine » R- a-methylhistamine = HTMT > imetit. To confirm the role of H)- receptors in the contractile response caused by histamine, I examined the effect two Hi- receptor selective antagonists, mepyramine and triprolidine on responses caused by this autacoid. Both mepyramine and triprolidine caused noncompetitive antagonism of the histamine contractions yielding apparent pA2 values of 9.4 ± 0.22 and 8.4 + 0.20, respectively (slopes of Schild Plots were significantly different from unity). To eliminate the potential interference of inhibitory H2-receptors in the contractile responses observed to histamine, I also examined the effect of the H2- receptor selective antagonist, tiotidine on responses produced by this autacoid. Tiotidine caused an increase in the maximum contractile response elicited by histamine confirming that inhibitory H2-receptors can interfere with the contractile responses induced by this autacoid in the isolated bovine iris. Consequently, I repeated experiments that investigated the antagonism of the histamine contractions by mepyramine in the presence of tiotidine. Once again, mepyramine caused noncompetitive antagonism of the histamine contractions in the presence of tiotidine yielding an apparent pA2 value of 7.1 + 0.06 (slope of Schild Plot was significantly different from unity). Both clobenpropit (H3-receptor antagonist) and thioperamide (H3- and H4- receptor antagonist) did not block contractions elicited by histamine in the bovine iris. These results strongly suggest that the Hi-receptors mediating contractile responses to histamine in the bovine iris are atypical and may represent a new subclass of histamine receptors. Under conditions of tone induced by the muscarinic agonist carbachol, histamine and H2-receptor selective agonists, amthamine and dimaprit caused concentration-related relaxations with the following rank order of potency: histamine > amthamine >> dimaprit. These results indicate that functional inhibitory histamine H2-receptors exists in the bovine iris. In conclusion, inhibitory prejunctional H^-receptors are present in the bovine iris. Both prejunctional H3- and a2-adrenoceptors coexist on sympathetic nerve terminal and may share a common pathway for inhibition of norepinephrine release. At postjunctional sites, prostanoids are involved in the contractile responses of the bovine iris to histamine. Both excitatory atypical Hi- receptors and inhibitory H2- receptors coexist in the bovine isolated iris. Pre- and postjunctional histamine receptors may play a role in the regulation of aqueous humor dynamics and IOP in the anterior uvea.
Creighton University
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