Human Ciliary Muscle Cells Research Articles

P.2.105 Zotepine in the treatment of chronic schizophrenia

The aims of the current studies were to determine the in vitro and in vivo ocular and non-ocular pharmacological properties of cabergoline using well documented receptor binding, cell-based functional assays, and in vivo models. Cabergoline bound to native and/or human cloned serotonin-2A/B/C (5HT2A/B/C), 5HT1A, 5HT7, α2B, and dopamine-2/3 (D2/3) receptor subtypes with nanomolar affinity. Cabergoline was an agonist at human recombinant 5HT2, 5HT1A and D2/3 receptors but an antagonist at 5HT7 and α2 receptors. In primary human ciliary muscle (h-CM) and trabecular meshwork (h-TM) cells, cabergoline stimulated phosphoinositide (PI) hydrolysis (EC50 = 19 ± 7 nM in TM; 76 nM in h-CM) and intracellular Ca2+ ([Ca2+]i) mobilization (EC50 = 570 ± 83 nM in h-TM; EC50 = 900 ± 320 nM in h-CM). Cabergoline-induced [Ca2+]i mobilization in h-TM and h-CM cells was potently antagonized by a 5HT2A-selective antagonist (M-100907, Ki = 0.29–0.53 nM). Cabergoline also stimulated [Ca2+]i mobilization more potently via human cloned 5HT2A (EC50 = 63.4 ± 10.3 nM) than via 5HT2B and 5HT2C receptors. In h-CM cells, cabergoline (1 μM) stimulated production of pro-matrix metalloproteinases-1 and -3 and synergized with forskolin to enhance cAMP production. Cabergoline (1 μM) perfused through anterior segments of porcine eyes caused a significant (27%) increase in outflow facility. Topically administered cabergoline (300–500 μg) in Dutch-belted rabbit eyes yielded 4.5 μM and 1.97 μM levels in the aqueous humor 30 min and 90 min post-dose but failed to modulate intraocular pressure (IOP). However, cabergoline was an efficacious IOP-lowering agent in normotensive Brown Norway rats (25% IOP decrease with 6 μg at 4 h post-dose) and in conscious ocular hypertensive cynomolgus monkeys (peak reduction of 30.6 ± 3.6% with 50 μg at 3 h post-dose; 30.4 ± 4.5% with 500 μg at 7 h post-dose). In ketamine-sedated monkeys, IOP was significantly lowered at 2.5 h after the second topical ocular dose (300 μg) of cabergoline by 23% (p < 0.02) and 35% (p < 0.004) in normotensive and ocular hypertensive eyes, respectively. In normotensive eyes, cabergoline increased uveoscleral outflow (0.69 ± 0.7 μL/min–1.61 ± 0.97 μL/min, n = 13; p < 0.01). However, only seven of the eleven ocular hypertensive monkeys showed significantly increased uveoscleral outflow. These data indicate that cabergoline's most prominent agonist activity involves activation of 5HT2, 5HT1A, and D2/3 receptors. Since 5HT1A agonists, 5HT7 antagonists, and α2 antagonists do not lower IOP in conscious ocular hypertensive monkeys, the 5HT2 and dopaminergic agonist activities of cabergoline probably mediated the IOP reduction observed with this compound in this species.

Endothelin-1 Stimulates the Release of Arachidonic Acid and Prostaglandins in Cultured Human Ciliary Muscle Cells: Activation of Phospholipase A2

In the present study we have examined the effects and mechanisms of endothelin-1 (ET-1) on arachidonic acid (AA) release and prostaglandin (PG) synthesis in human ciliary muscle (HCM) cells. ET-1 stimulated AA release in a time (t1/2=1.5 min) and concentration-dependent (EC50=5 nM) manner, which is primarily mediated through the ETAreceptor subtype. The AA liberated by ET-1 appears to derive mainly from the phosphoinositides and phosphatidylcholine. Our data show that phospholipase A2(PLA2), but not phospholipase C (PLC), plays an important role in ET-1-induced AA release. This conclusion is supported by the following findings: (1) ET-1-evoked AA release was inhibited by the PLA2inhibitors dexamethasone, mepacrine and manoalide in a concentration-dependent manner. Conversion of AA into PGE2was inhibited by the cyclooxygenase inhibitors in the following order: Indomethacin>naproxen >ibuprofen>NS-398>aspirin. (2) The phorbol ester, PDBu, an activator of protein kinase C, potentiated ET-1-induced AA release by 39%, but inhibited that of inositol phosphates formation by 62%. (3) Pretreatment of the labeled cells with isoproterenol lowered ET-1-induced inositol phosphates production, but had no effect on AA release. (4) U71322, a PLC inhibitor, inhibited ET-1-induced inositol phosphates production, but had no effect on that of AA release. (5) Pretreatment of the cells with pertussis toxin (0.1 μg ml−1) attenuated the stimulatory effects of ET-1 on AA release and PGE2formation. These data demonstrate that ET-1 is a potent agonist for AA release and PG synthesis in HCM cells, and that PLA2, but not PLC, plays an important role in ET-1-induced AA release and PG synthesis. In ciliary muscle, AA and its metabolites play important roles in intracellular signalling, modulation of physiological processes, and regulation of intraocular pressure.

Detection of EP 2, EP 4, and FP receptors in human ciliary epithelial and ciliary muscle cells

We have examined the expression of three prostaglandin (PG) receptors, EP 2, EP 4, and FP, in a nonpigmented ciliary epithelial cell line (ODMC1-2) and in human ciliary muscle (HCM) cells. Total RNA preparations from either ODMCl-2 or HCM cells were subjected to reverse transcription-polymerase chain reaction (RT-PCR) with sense and antisense primers for each of the three PG receptors. The RT-PCR generated DNA products of predicted sizes corresponding to the EP 2, EP 4, and FP receptors in both ODMCl-2 and HCM cells. PCR products corresponding to each receptor were hybridized with specific 32P-labeled probes and, for further confirmation, digested with appropriate restriction enzymes. Pharmacological studies with the EP 2 receptor-selective agonist butaprost resulted in a significant increase in the cyclic AMP level in ODMCl-2 cells. The stimulation of cyclic AMP in ODMCl-2 cells by PGE 2 and 11-deoxy PGE 1, the respective EP 1/EP 2/EP 3 EP4 and EP 2/EP 3 EP 4 receptor agonists, was concentration-dependently inhibited by the EP 4 receptor-selective antagonist AH23848. These results conclusively demonstrate the presence of both mRNA and protein for EP 2, EP 4, and FP receptors in ODMC1-2 and HCM cells.

Histamine Induced Contraction of Human Ciliary Muscle Cells

In cultured human ciliary muscle cells we previously showed that histamine, via an H1receptor, stimulates the production of inositol phosphates and mobilization of intracellular calcium. We further investigated in this study whether histamine would cause contraction of human ciliary muscle cells. Photomicrographs were taken of the ciliary muscle cells before and after exposure to histamine. Cross sectional surface area of the cells was quantified using image analysis software. A decrease in cross sectional surface area was interpreted as an indication of cell contraction. The results of this study indicated that histamine (10−6M–10−4M) caused contraction of human ciliary muscle cells in a concentration-dependent fashion. The effect of histamine was mediated by the H1receptor subtype since the histamine effect was antagonized by 10−6Mchlorpheniramine (an H1receptor subtype selective antagonist) but not by 10−4Mcimetidine (H2antagonist) or thioperamide (H3antagonist). The phospholipase C (PLC) inhibitor, U73122 (10−6M) and the intracellular calcium store depleting agent thapsigargin (10−6M) both prevented the histamine induced contraction, demonstrating that the activation of PLC and the intracellular calcium release were the key steps necessary for contraction. Our data indicate that in ciliary muscle cells, histamine, via an H1receptor, activates PLC and increases intracellular calcium, which subsequently causes contraction of the cells.

Effects of exogenous prostaglandins on aqueous humor dynamics and blood-aqueous barrier function

Topical prostaglandins (PGs) are very effective at reducing intraocular pressure (IOP) in a variety of animals and in humans with relatively few side effects. The mechanisms of action of several PGs, their prodrugs and analogues have been studied in rabbits, cats, monkeys and humans. PGF 2α and its analogues evaluated in monkeys include PGF 2α-tromethamine salt, PGF 2α-isopropylester (-IE), S-1033, PhXA34, PhDH100A and latanoprost (PhXA41). Aqueous flow and outflow facility are either increased or remain unchanged by these agents. PGF 2α-IE, PHXA34, PhDH100A and latanoprost increase uveoscleral outflow, accounting for most of the IOP reduction. PGA 2 in cats increases aqueous flow and outflow facility, but it reduces IOP primarily by stimulating uveoscleral outflow. The PGD 2 analogue BW245C is unique in that it is the only PG that decreases aqueous flow. Mechanistic studies in humans have been performed with PGF 2α-IE, unoprostone, PhXA34 and latanoprost. In two clinical studies with latanoprost, a significant increase in uveoscleral outflow was found which, as in animals, accounts for most of the IOP reduction. A slight but inconsistent increase in outflow facility may also be involved, The doses tested had minimal effects on the permeability of the blood-aqueous barrier (BAB). In vitro studies of human tissue have been conducted to elucidate the PG effect on outflow facility and uveoscleral outflow. Studies of isolated human anterior segment preparations show that PGE 2 increases outflow facility whereas PGF 2α, has no measurable effect on this parameter. Studies of human ciliary muscle cells in tissue culture indicate that PGs may directly modulate extracellular matrix metabolism, which may be related to the increased uveoscleral drainage. This review summarizes in vitro and in vivo studies of the effects of PGs on aqueous humor dynamics and BAB integrity in humans, cats and monkeys.

SHORT COMMUNICATION Intracellular calcium mobilization following prostaglandin receptor activation in human ciliary muscle cells

PURPOSE. To investigate the presence of specific prostaglandin receptors in primary cultures of human ciliary muscle cells by measuring agonist-stimulated intracellular calcium mobilization. METHODS. The ciliary muscle cells, cultured from postmortem human ciliary muscle explants, were first characterized by anti-desmin and anti-smooth muscle a-actin antibodies. Increase in intracellular calcium concentrations, in fura 2-AM loaded human ciliary muscle cells stimulated by prostaglandin receptor agonists, were determined with a digital fluorescence imaging system. RESULTS. The resting intracellular calcium concentration in the fura 2-AM loaded cells was 60.0 ± 6.0 nM. The threshold concentration of PG receptor agonists needed to increase the concentration of intracellular calcium was 10 - 8 M. The stimulation of these cells by PGF 2a, 17-phenyl trinor PGE 2, and U46619, the FP, EP 1, and TP receptor agonists, resulted in the dose-dependent increase of intracellular calcium. CONCLUSION. The results of the present study suggest that EP 1, FP, and TP receptors are present in human ciliary muscle cells.

Effect of Histamine on Phosphoinositide Turnover and Intracellular Calcium in Human Ciliary Muscle Cells

This report describes the effect of histamine on phospholipase C (PLC) activity and calcium mobilization in cultured human ciliary muscle cells. PLC activity was assessed by measuring the production of inositol phosphates and intracellular calcium mobilization was assessed by Fura 2 ratio fluorometry. The stimulation of PLC by histamine was concentration dependent with an EC 50of 0.96μ m. The H 1antagonist chlorpheniramine blocked the response with an IC 50of 0.53μ m. Calcium fluorometry experiments indicated a mean basal calcium concentration of 36n mwith a 10 -4 mhistamine induced mean peak value of 1132n mfollowed by a gradually declining plateau phase. EC 50and IC 50(chlorpheniramine) values from histamine induced peak calcium concentrations agreed with the PLC results. Pretreatment of the cells with the PLC inhibitor U73122 at 10 -6 mcompletely blocked histamine induced calcium mobilization. Removal of extracellular calcium eliminated the plateau phase but not the initial calcium peak indicating that both intra and extracellular calcium sources are required for a normal response. The calcium ATPase inhibitor thapsigargin caused depletion of intracellular calcium stores and prevented a subsequent normal calcium mobilization response to histamine. Ryanodine, a release inhibitor of certain intracellular calcium stores, had no effect on the histamine induced response. The results of these experiments indicate that histamine, via an H 1receptor, activated the PLC second messenger pathway, and caused a multi-phasic mobilization of both intracellular and extracellular calcium. The entry of the extracellular calcium was shown to be dependent upon release of calcium from a ryanodine insensitive intracellular store.

Global/local task indices of hemispheric and attentional functions in schizophrenia

The aims of the current studies were to determine the in vitro and in vivo ocular and non-ocular pharmacological properties of cabergoline using well documented receptor binding, cell-based functional assays, and in vivo models. Cabergoline bound to native and/or human cloned serotonin-2A/B/C (5HT2A/B/C), 5HT1A, 5HT7, α2B, and dopamine-2/3 (D2/3) receptor subtypes with nanomolar affinity. Cabergoline was an agonist at human recombinant 5HT2, 5HT1A and D2/3 receptors but an antagonist at 5HT7 and α2 receptors. In primary human ciliary muscle (h-CM) and trabecular meshwork (h-TM) cells, cabergoline stimulated phosphoinositide (PI) hydrolysis (EC50 = 19 ± 7 nM in TM; 76 nM in h-CM) and intracellular Ca2+ ([Ca2+]i) mobilization (EC50 = 570 ± 83 nM in h-TM; EC50 = 900 ± 320 nM in h-CM). Cabergoline-induced [Ca2+]i mobilization in h-TM and h-CM cells was potently antagonized by a 5HT2A-selective antagonist (M-100907, Ki = 0.29–0.53 nM). Cabergoline also stimulated [Ca2+]i mobilization more potently via human cloned 5HT2A (EC50 = 63.4 ± 10.3 nM) than via 5HT2B and 5HT2C receptors. In h-CM cells, cabergoline (1 μM) stimulated production of pro-matrix metalloproteinases-1 and -3 and synergized with forskolin to enhance cAMP production. Cabergoline (1 μM) perfused through anterior segments of porcine eyes caused a significant (27%) increase in outflow facility. Topically administered cabergoline (300–500 μg) in Dutch-belted rabbit eyes yielded 4.5 μM and 1.97 μM levels in the aqueous humor 30 min and 90 min post-dose but failed to modulate intraocular pressure (IOP). However, cabergoline was an efficacious IOP-lowering agent in normotensive Brown Norway rats (25% IOP decrease with 6 μg at 4 h post-dose) and in conscious ocular hypertensive cynomolgus monkeys (peak reduction of 30.6 ± 3.6% with 50 μg at 3 h post-dose; 30.4 ± 4.5% with 500 μg at 7 h post-dose). In ketamine-sedated monkeys, IOP was significantly lowered at 2.5 h after the second topical ocular dose (300 μg) of cabergoline by 23% (p < 0.02) and 35% (p < 0.004) in normotensive and ocular hypertensive eyes, respectively. In normotensive eyes, cabergoline increased uveoscleral outflow (0.69 ± 0.7 μL/min–1.61 ± 0.97 μL/min, n = 13; p < 0.01). However, only seven of the eleven ocular hypertensive monkeys showed significantly increased uveoscleral outflow. These data indicate that cabergoline's most prominent agonist activity involves activation of 5HT2, 5HT1A, and D2/3 receptors. Since 5HT1A agonists, 5HT7 antagonists, and α2 antagonists do not lower IOP in conscious ocular hypertensive monkeys, the 5HT2 and dopaminergic agonist activities of cabergoline probably mediated the IOP reduction observed with this compound in this species.

Prostaglandins increase proMMP-1 and proMMP-3 secretion by human ciliary smooth muscle cells.

The mechanism by which prostaglandin(PG)F2 alpha increases uveoscleral outflow and lowers intraocular pressure in primates is not known. In cultured human ciliary muscle cells, PGF2 alpha induces the expression of the protooncogene c-fos which is known to induce the transcription of genes such as matrix metalloproteinase-1 (MMP-1) and MMP-3 in other cell systems. As these enzymes are initially secreted as proenzymes, the present study was undertaken to determine if PG treatment induces ciliary muscle cells to secrete either proMMP-1 or proMMP-3. Human ciliary smooth muscle cells were grown to confluence in monolayer cell cultures and then treated with PGF2 alpha, 17-phenyltrinor-PGF2 alpha, or 11-deoxy-PGE1. Medium harvested at various times after treatment was assayed for proMMP-1 and proMMP-3 content using sandwich ELISAs. Three days after adding 10 nM PGF2 alpha, proMMP-1 and proMMP-3, concentrations in the culture medium were increased by 254 +/- 33% (mean +/- SE) and 128 +/- 13%, respectively. Compared with vehicle controls, 24 h treatment with 200 nM PGF2 alpha, 17-phenyltrinor-PGF2 alpha, or PGE1, increased proMMP-1 by 116 +/- 29%, 169 +/- 26%, and 273 +/- 16%, respectively. In parallel experiments, proMMP-3 was increased by 99 +/- 18%, 82 +/- 24%, and 214 +/- 16%, respectively. These results suggest that induction of MMPs in situ following topical PG treatment may degrade ciliary muscle extracellular matrix and possibly contribute to increased uveoscleral outflow, as well.

Characterization of Muscarinic Receptor Involvement in Human Ciliary Muscle Cell Function

Muscarinic agonist-induced contraction of the ciliary muscle is generally believed to increase aqueous outflow facility and effect accommodation. We used cultured human ciliary muscle cells as a model to study the muscarinic receptor subtype(s) involved in the contractile response of the muscle. Thus, a single cell contraction assay for these muscle cells was developed. And since agonist-induced contraction of smooth muscles is expected to involve the activation of phospholipase C (PLC), we also monitored the PLC activity in these cells. Carbachol caused contraction of the muscle cells in a dose-dependent and time-dependent manner with an estimated EC50 of 1-3 microM. The contractile effect of 100 microM carbachol was antagonized by pretreatment of atropine (1 microM) and 4DAMP (10 nM, antagonist selective for the M1 and M3 receptors) but not by pirenzepine (10 microM, antagonist selective for the M1 receptor), suggesting the involvement of the M3 but not the M1 muscarinic receptor. M3 receptor is also essential for the carbachol-induced PLC activation in the ciliary muscle cells, as indicated by the activity profiles of receptor subtype selective antagonists. For example, the stimulative effect of carbachol (EC50 = 20 microM) was antagonized by pirenzepine (pKi = 6.8), HHSiD (pKi = 7.6), 4DAMP (pKi = 9.5) and methoctramine (pKi < 6). Thus, these results indicate that an M3-like receptor subtype is essential in mediating the muscarinic agonists-induced functional changes, such as PLC activation or muscle contraction, in the ciliary muscle.

Characterization of Muscarinic Receptors in Cultured Human Iris Sphincter and Ciliary Smooth Muscle Cells

Muscarinic receptors present in cultured human iris sphincter and ciliary smooth muscle cells were characterized by both ligand ([ 3H]QNB binding) and functional (phosphoinositide hydrolysis) studies. Ligand binding studies showed that [ 3H]QNB represented a single population of binding sites with K D values of 4·02 × 10 -11 M in the ciliary and 5·6 × 10 -11 M in the iris sphincter cells. In competition studies, the selective antagonist, 4-diphenylacetoxy- N-methylpiperidine-methobromide (4-DAMP) was the most potent in displacing [ 3H]QNB with selectivity of 150-350-fold over pirenzepine (M 1) and 450-1700-fold over AF-DX 116 (M 2). 4-DAMP recognized one site in the iris sphincter cells ( K i = 0·34 nM) but two sites in the ciliary cells ( K H = 0·9 nM and K L= 49 nM). 4-DAMP was also the most potent in inhibiting carbachol-induced hydrolysis of inositol phospholipids (PI) in both cell types. However, the IC 50 values for PI hydrolysis were several fold lower than those for [ 3H]QNB binding. Using these selective antagonists, our data supports the presence of functional muscarinic receptors of M 3 subtype in human iris sphincter and ciliary cells. It also shows the presence of a second low affinity site in the ciliary smooth muscle cells that is recognized by 4-DAMP.

Characterization of acetylcholine- and endothelin-induced calcium entry in cultured human ciliary muscle cells.

We characterized the effects of acetylcholine and endothelin on cultured human ciliary muscle cells, using the calcium-sensitive dye fura-2 to measure intracellular calcium and intracellular microelectrodes to measure the membrane potential. Both agonists, endothelin and acetylcholine, had a typical biphasic effect on the intracellular calcium concentration. Calcium peaked initially, because of its release from intracellular stores, and then reached a plateau, owing to entry of extracellular calcium. Endothelin-induced calcium entry was almost completely blocked by addition of extracellular La3+ (50 mumol/l) and Ni2+ (1 mmol/l). Acetylcholine-induced calcium entry was likewise almost completely abolished by La3+ and Ni2+. Both endothelin and acetylcholine led to an initial transient hyperpolarization with a subsequent depolarization. The hyperpolarization of the membrane potential had a time course similar to the initial calcium peak, while the depolarization occurred parallel to the calcium plateau. The depolarization induced by both agonists was reduced in the presence of La3+ and Ni2+. Verapamil (10 mumol/l) had no effect on either the calcium entry or the depolarization. Acetylcholine did not induce a [Ca2+]i peak when it was applied during the endothelin-induced [Ca2+]i plateau and vice versa. The [Ca2+]i plateau was not higher with concomitant than with single application of acetylcholine or endothelin. Thus, calcium entry and membrane depolarization induced by acetylcholine and endothelin seem to be mediated by a common La(3+)- and Ni(2+)-sensitive but verapamil-insensitive mechanism.

Regulation of intracellular calcium in cultured human ciliary muscle cells

Regulation of intracellular calcium in cultured human ciliary muscle cells

Na(+)-dependent HCO3- transport and Na+/H+ exchange regulate pHi in human ciliary muscle cells.

We investigated intracellular pH (pHi) regulation in cultured human ciliary muscle cells by means of the pH-sensitive absorbance of 5(and 6)-carboxy-4',5'-dimethylfluorescein (CDMF). The steady-state pHi was 7.09 +/- 0.04 (n = 12) in CO2/HCO3(-)-buffered and 6.86 +/- 0.03 (n = 12) in HEPES-buffered solution. Removal of extracellular sodium for 6 min acidified the cells by 1.11 +/- 0.06 pH units (n = 12) in the presence of CO2/HCO3- and by 0.91 +/- 0.05 pH units (n = 8) in its absence. Readdition of external sodium resulted in a rapid pHi recovery, which was almost completely amiloride-sensitive in the absence of CO2/HCO3- but only slightly influenced by amiloride in its presence. Application of DIDS under steady-state conditions significantly acidified the ciliary muscle cells by 0.25 +/- 0.02 (n = 4) in 6 min, while amiloride had no effect. The pHi recovery after an intracellular acid load was completely dependent on extracellular sodium. In HEPES-buffered solution the pHi recovery was almost completely mediated by Na+/H+ exchange, since it was blocked by amiloride (1 mmol/liter). In contrast, a marked amiloride-insensitive pHi recovery was observed in CO2/HCO3(-)-buffered solution which was mediated by chloride-independent and chloride-dependent Na+ HCO3- cotransport. This recovery, inhibited by DIDS (0.2 mmol/liter), was also observed if the cells were preincubated in chloride-free solution for 4 hr. Analysis of the sodium dependence of the pHi recovery after NH4Cl prepulse revealed Vmax = 0.57 pH units/min, Km = 39.7 mmol/liter extracellular sodium for the amiloride-sensitive component and Vmax = 0.19 pH units/min, Km = 14.3 mmol/liter extracellular sodium for the amiloride-insensitive component. We conclude that Na+/H+ exchange and chloride-independent and chloride-dependent Na(+)-HCO3- cotransport are involved in the pHi regulation of cultured human ciliary muscle cells.

Electrogenic sodium-bicarbonate cotransport in human ciliary muscle cells.

We investigated membrane voltage and intracellular pH (pHi) in cultured human ciliary muscle cells using a cell line (H7CM) and primary-cultured human ciliary muscle cells. 1) Resting potential was 58.9 +/- 1.0 mV in H7CM cells and 61.9 +/- 1.4 mV in primary cultures. The following data are from H7CM cells, but results from primary cultures were basically similar. 2) In HCO3(-)-CO2-buffered solution, removal of extracellular sodium resulted in a depolarization [change in membrane resistance (delta V) = 31.3 +/- 2.8 mV] that was less marked in the absence of HCO3(-)-CO2 (delta V = 0.5 +/- 2.6 mV) and reduced by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (delta V = 19.3 +/- 1.9 mV). 3) Removal of extracellular HCO3(-)-CO2 led to a depolarization (delta V = 13.2 +/- 0.8 mV) that was abolished in the absence of extracellular sodium and inhibited by DIDS. 4) Intracellular alkalinization led to a depolarization (delta V = 24.7 +/- 2.3 mV), and intracellular acidification resulted in a hyperpolarization (delta V = 9.4 +/- 1.1 mV) that was inhibited by DIDS and dependent on extracellular HCO3(-)-CO2 and sodium. 5) pHi backregulation after an acid load occurred in both the presence and absence of extracellular bicarbonate but not in the absence of extracellular sodium. Our data are consistent with an electrogenic Na(+)-HCO3- cotransport in human ciliary muscle cells, which is activated by intracellular acidification.

Synthesis of extracellular matrix components by human ciliary muscle cells in culture.

The production and spatial organization of connective tissue components in ciliary muscle cell cultures was studied with immunohistochemical and ultrastructural methods. Antibodies against collagen types IV and VI, fibronectin and laminin were used. Laminin stains as pericellular network surrounding individual muscle cells. Type IV collagen shows positive cytoplasmic staining and only small foci of extracellular immunofluorescence. Staining for type VI collagen and fibronectin is seen near the ends of the bipolar cells, while the lateral sides of the cells remain unstained. Electronmicroscopy shows that cultured ciliary muscle cells are surrounded by an incomplete basal lamina. In addition, bundles of 5-20 nm thick extracellular microfibrils are seen. The bundles are oriented parallel to the axis of the cells and are in close contact with the cell membrane in areas where membrane-bound dense bands are formed. Immunoelectronmicroscopy indicates that the bundles contain fibronectin and type VI collagen fibrils. While the fibronectin fibrils approach the cell membrane directly, type VI collagen fibrils are usually separated from the cell membrane by fine fibrillous material of different nature. Quality and spatial organization of the extracellular material in ciliary muscle cell cultures shows marked similarities with the extracellular matrix of ciliary muscle in situ.

Endothelin depolarizes membrane voltage and increases intracellular calcium concentration in human ciliary muscle cells

The ciliary muscle which is involved in accomodation and regulation of aqueous humour outflow resistance resembles smooth muscle in other parts of the body. In the present investigation we used an established primary cell line (H7CM) to study the effects of endothelin, a novel vasoconstrictor peptide, on membrane voltage (V) and intracellular calcium in cultured human ciliary muscle cells. Membrane voltage was measured in confluent monolayers of H7CM cells using conventional microelectrodes. Intracellular calcium concentration ([Ca] i) was measured in single H7CM cells using the fluorescent calcium indicator fura-2. Under resting conditions V averaged −66.9 ± 0.7 mV (mean ± SEM, n=125). Endothelin (10 −10–10 −6M) induced a dose-dependent reversible membrane voltage depolarization and a dose-dependent rise in [Ca] i. The initial calcium peak was followed by a recovery phase during which oscillations of [Ca] i occurred. The initial calcium peak was not dependent on the presence of extracellular calcium and was not abolished in the presence of the calcium antagonist verapamil (10 −4M). Thus it is probably mediated by a release of calcium from intracellular reservoirs. We conclude that cultured human ciliary muscle cells express a functional endothelin receptor.