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

The ciliary muscle which is involved in accommodation 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(-6)M) 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(-4)M). 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.     

Stimulation of ciliary beat frequency by autonomic agonists: in vivo

beta 2-Adrenergic bronchodilator and muscarinic cholinergic bronchoconstrictor agonists both stimulate ciliary activity in vitro. To test the hypothesis that increases in autonomic activity would result in increases in ciliary beat frequency (CBF) in vivo, a correlation analysis heterodyne laser light-scattering system was developed and validated to measure the stimulating effects of sympathomimetic and parasympathomimetic agonists on tracheal CBF in intact, anesthetized beagles. The mean baseline CBF from 42 studies of 274 measurements in 9 (5 male and 4 female) adult beagles was 6.6 +/- 1.1 Hz. The stimulating effects of a beta 2-adrenergic agonist, fenoterol, and a muscarinic cholinergic agonist, methacholine, on CBF were studied on four and eight beagles, respectively. The studies were randomized and blinded. Aerosolized 10(-5) M fenoterol stimulated the CBF from the base line of 6.8 +/- 2.5 to 32.0 +/- 17.9 Hz in four dogs. Aerosolized methacholine stimulated the CBF from the base line of 5.8 +/- 0.7 to 9.4 +/- 3.0 Hz for 10(-8) M, and to 12.6 +/- 3.1 Hz for 10(-6) M in eight dogs. These are the first data obtained in intact animals that demonstrate CBF in the lower respiratory tract is regulated by autonomic agonists.     

Effect of cAMP on ciliary function in rabbit tracheal epithelial cells

To study the effect of adenosine 3',5'-cyclic monophosphate (cAMP) on respiratory ciliary activity, we measured ciliary beat frequency (CBF) of rabbit tracheal epithelium by a photoelectric method in response to cAMP analogues and agents that can increase endogenous cAMP production. Addition of 8-bromo-cAMP dose dependently enhanced CBF, with the maximal increase and the concentration necessary to produce a half-maximal response (KD) being 31.0 +/- 3.4% (SE) (P less than 0.001) and 3.2 +/- 1.5 x 10(-7) M, respectively. Other structurally dissimilar cAMP analogues dibutyryl cAMP and chlorophenylthio-cAMP likewise caused increases in CBF. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and the adenylate cyclase stimulator forskolin also augmented CBF in a dose-dependent fashion and were accompanied by the increases in intracellular concentrations of cAMP. Ciliary discoordination was not observed in any of the experiments. These results suggest that cAMP may accelerate mucociliary clearance through the activation of ciliary motility and that intracellular cAMP levels appear to be an important determinant for the lung mucociliary transport functions.     

Thromboxane A2 mimetic U46619 stimulates ciliary motility of rabbit tracheal epithelial cells.

To elucidate whether thromboxane A2 (TxA2), one of the important arachidonic acid metabolites that may play a role in the development of airway inflammation, affects respiratory ciliary motility and, if so, what the mechanism of action is, we measured ciliary beat frequency (CBF) of rabbit cultured tracheal epithelium in response to U46619, a TxA2 mimetic agonist, by a photoelectric method. Addition of U46619 (10(-5) M) increased CBF from 17.7 +/- 0.7 to 22.8 +/- 1.4 Hz (mean +/- SE, p less than 0.01) within 5 min, which was followed by a decline to the baseline value by 10 min. This effect was concentration-dependent, the maximal increase from the baseline value and the drug concentration required to produce a half-maximal effect (EC50) being 26.9 +/- 4.6% (p less than 0.01) and 3 x 10(-7) M, respectively. The U46619-induced increase in CBF was abolished by SQ29548, and TxA2 receptor antagonist, and inhibited by verapamil, a Ca(2+)-entry blocker, and H-7, a protein kinase C inhibitor. These results suggest that TxA2 stimulates ciliary motility through the activation of airway epithelial TxA2 receptors, and that this effect may be exerted from Ca(2+)-influx and protein kinase C.     

Vasoactive intestinal peptide stimulates ciliary motility in rabbit tracheal epithelium: modulation by neutral endopeptidase.

We studied the effect of vasoactive intestinal peptide (VIP) on ciliary activity in rabbit cultured tracheal epithelium by a photoelectric method in vitro. Administration of VIP (10(-7) M) elicited an increase in ciliary beat frequency (CBF) from the baseline values of 970 +/- 52 to 1139 +/- 75 beats/min (mean +/- S.E., P less than 0.01). This ciliostimulatory effect was dose-dependent, with the maximal increase and EC50 value being 17.4 +/- 1.0% (P less than 0.05) and 6.10(-11) M, respectively. The VIP-induced increase in CBF was abolished by pretreatment of cells with [4-Cl-D-Phe6, Leu17]-VIP, a VIP receptor antagonist. The neutral endopeptidase inhibitor phosphoramidon (10(-5) M) potentiated the effect of VIP, so that the CBF dose-response curve for VIP was shifted to lower concentrations by 0.5 log U. The administration of VIP increased cyclic AMP levels in epithelial cells, an effect that was also potentiated by phosphoramidon. These results suggest that VIP may interact with its specific receptors and stimulate airway ciliary activity probably through the activation of adenylate cyclase, and that neutral endopeptidase may play a role in modulating this effect of VIP.     

Differential effects of UTP, ATP, and adenosine on ciliary activity of human nasal epithelial cells

The purinergic regulation of ciliary activity was studied using small, continuously superfused explants of human nasal epithelium. The P2Y(2) purinoceptor (P2Y(2)-R) was identified as the major purinoceptor regulating ciliary beat frequency (CBF); UTP (EC(50) = 4.7 microM), ATP, and adenosine-5'-O-(3-thiotriphosphate) elicited similar maximal responses, approximately twofold over baseline. ATP, however, elicited a post-peak sustained plateau in CBF (1.83 +/- 0.1-fold), whereas the post-peak CBF response to UTP declined over 15 min to a low-level plateau (1.36 +/- 0.16-fold). UDP also stimulated ciliary beating, probably via P2Y(6)-R, with a maximal effect approximately one-half that elicited by P2Y(2)-R stimulation. Not indicated were P2Y(1)-R-, P2Y(4)-R-, or P2Y(11)-R-mediated effects. A(2B)-receptor agonists elicited sustained responses in CBF approximately equal to those from UTP/ATP [5'-(N-ethylcarboxamido)adenosine, EC(50) = 0.09 microM; adenosine, EC(50) = 0.7 microM]. Surprisingly, ADP elicited a sustained stimulation in CBF. The ADP effect and the post-peak sustained portion of the ATP response in CBF were inhibited by the A(2)-R antagonist 8-(p-sulfophenyl)theophylline. Hence, ATP affects ciliary activity through P2Y(2)-R and, after an apparent ectohydrolysis to adenosine, through A(2B)AR.     

Phorbol ester contracts rabbit thoracic aorta by increasing intracellular calcium and by activating calcium influx

The ability of the phorbol ester tumor promoter, PDB, to activate contraction and stimulate calcium influx was investigated in rabbit thoracic aorta. PDB caused a strong, slowly-developing sustained contraction in physiological salt solution which was concentration-related (0.01 to 10.0 microM). PDB-induced contractions (0.1 microM) in calcium-free medium were attenuated but not prevented. PDB (1.0 microM) maximally stimulated Ca influx above basal control, vehicle = 39.2 +/- 2.2; PDB 1.0 microM = 70.7 +/- 6.7 mumoles Ca/kg tissue; N = 16, p less than 0.01). These data suggest that PDB activates rabbit thoracic aorta by a combination of intracellular and extracellular calcium dependent mechanisms.     

Pathways of substance P stimulation of canine tracheal ciliary beat frequency.

Substance P (SP), an inflammatory neuropeptide, may be released by intraepithelial nerves in response to an irritant or inflammatory stimulus. To investigate the neural and humoral pathways mediating the response of tracheal ciliary beat frequency (CBF) to topically applied SP, CBF was measured on the ventral midtracheal surface of anesthetized beagles by using heterodyne-mode correlation analysis laser light scattering. In the first study, aerosolized SP, delivered to the lungs of eight beagle dogs, stimulated CBF in a dose-dependent manner from a baseline of 4.9 +/- 0.4 Hz to a maximum of 14.9 +/- 1.5 Hz at dose of 10(-7) M. In the second study, the tracheal lumen was isolated from the bronchial airways by inflating the cuff of an endotracheal tube near the carina. Intravenous hexamethonium bromide (2 mg/kg), ipratropium bromide (0.5 micrograms/kg), and indomethacin (2 mg/kg) were used as blocking agents to inhibit the nicotinic, muscarinic, and cyclooxygenase pathways, respectively. Aerosolized 10(-9), 10(-8), or 10(-7) M SP was delivered sequentially to the tracheal lumen for 3 min at 30-min intervals. SP caused two distinct CBF stimulatory episodes at 4 min (mean time of the maximal response) and at 18 min (mean time of the maximal response) after onset of delivery and returned to baseline after 25 min. SP stimulated CBF from the baseline of 5.1 +/- 0.4 Hz to a maximum of 14.2 +/- 2.5 Hz during the first episode (P less than 0.01) and to 10.4 +/- 0.6 Hz during the second episode (P less than 0.01) at dose of 10(-8) M. These responses were inhibited by all the blocking agents. These data suggest that SP stimulates CBF via a cyclooxygenase-dependent parasympathetic reflex.     

Regulation of ciliary beat frequency by autonomic mechanisms: in vitro

The ciliated epithelium of the mammalian trachea separates the neurohumoral milieu of the tissue from that of the environment of the airway lumen. To determine whether specific autonomic receptors regulating ciliary beat frequency (CBF) were located on mucosal or serosal sides, we measured CBF by heterodyne mode correlation analysis laser light scattering in bovine tracheal tissues mounted in a two-sided chamber. A beta 2-adrenergic agonist, fenoterol, at 10(-7) M, stimulated serosal CBF from 7.9 +/- 1.3 to 20.2 +/- 5.8 Hz (P less than 0.01) and mucosal CBF from 6.6 +/- 0.9 to 14.7 +/- 4.6 Hz (P less than 0.01). A muscarinic cholinergic agonist, methacholine, at 10(-7) M, increased mucosal CBF from 8.4 +/- 1.0 to 19.5 +/- 5.5 Hz (P less than 0.01) and serosal CBF from 8.0 +/- 0.9 to 15.4 +/- 5.0 Hz (P less than 0.01). The differences in stimulation of CBF on the mucosal and serosal sides between fenoterol and methacholine were significant (P less than 0.01). Studies in which these autonomic agonist stimulating effects were inhibited by their respective antagonists, propranolol and atropine sulfate, demonstrated that CBF can be regulated independently by mediators both in the submucosa and within the mucus lining.     

Signal Transduction in Smooth Muscle: Selected Contribution: Airway caliber in healthy and asthmatic subjects: effects of bronchial challenge and deep inspirations

In 9 healthy and 14 asthmatic subjects before and after astandard bronchial challenge and a modified [deep inspiration (DI), inhibited] bronchial challenge and after albuterol, we tracked airwaycaliber by synthesizing a method to measure airway resistance (Raw;i.e., lung resistance at 8 Hz) in real time. We determined the minimumRaw achievable during a DI to total lung capacity and the subsequentdynamics of Raw after exhalation and resumption of tidal breathing.Results showed that even after a bronchial challenge healthy subjectscan dilate airways maximally, and the dilation caused by a single DItakes several breaths to return to baseline. In contrast, at baseline,asthmatic subjects cannot maximally dilate their airways, and thisworsens considerably postconstriction. Moreover, after a DI, thedilation that does occur in airway caliber in asthmatic subjectsconstricts back to baseline much faster (often after a single breath).After albuterol, asthmatic subjects could dilate airways much closer tolevels of those of healthy subjects. These data suggest that theasthmatic smooth muscle resides in a stiffer biological state comparedwith the stimulated healthy smooth muscle, and inhibiting a DI inhealthy subjects cannot mimic this.

     

Selected contribution: airway caliber in healthy and asthmatic subjects: effects of bronchial challenge and deep inspirations.

In 9 healthy and 14 asthmatic subjects before and after a standard bronchial challenge and a modified [deep inspiration (DI), inhibited] bronchial challenge and after albuterol, we tracked airway caliber by synthesizing a method to measure airway resistance (Raw; i.e., lung resistance at 8 Hz) in real time. We determined the minimum Raw achievable during a DI to total lung capacity and the subsequent dynamics of Raw after exhalation and resumption of tidal breathing. Results showed that even after a bronchial challenge healthy subjects can dilate airways maximally, and the dilation caused by a single DI takes several breaths to return to baseline. In contrast, at baseline, asthmatic subjects cannot maximally dilate their airways, and this worsens considerably postconstriction. Moreover, after a DI, the dilation that does occur in airway caliber in asthmatic subjects constricts back to baseline much faster (often after a single breath). After albuterol, asthmatic subjects could dilate airways much closer to levels of those of healthy subjects. These data suggest that the asthmatic smooth muscle resides in a stiffer biological state compared with the stimulated healthy smooth muscle, and inhibiting a DI in healthy subjects cannot mimic this.     

Thapsigargin increases cytoplasmic free Ca2+ without influencing steroidogenesis in chicken granulosa cells.

The effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) and progesterone production were determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye Fura-2. Thapsigargin stimulated a 4.6 +/- 0.2-fold increase in [Ca2+]i from a resting level of 55 +/- 6 nM up to 233 +/- 23 nM (n = 8) in 100% of the cells tested (n = 86). However, two different response patterns were observed. Dependent on the cell populations, a maximally effective concentration of thapsigargin (100 nM) stimulated either a rapid (within 16 +/- 2 s) transient increase in [Ca2+]i or a slowly (99 +/- 20 s) developing and sustained increase in [Ca2+]i. Both [Ca2+]i responses were concentration (0.001-1 microM)-dependent with an EC50 around 40 nM. The transient [Ca2+]i response occurred in the absence of extracellular Ca2+ and was unaffected by pretreating the cells with the Ca2+ channel blockers methoxyverapamil (50 microM) or lanthanum (1 mM). The plateau phase of the sustained [Ca2+]i response returned to resting level in the absence of extracellular Ca2+, but remained elevated in the presence of methoxyverapamil (50 microM) or lanthanum (1 mM). Despite its ability to cause transient or prolonged increases in [Ca2+]i, thapsigargin (0.001-1 microM) did not affect basal or luteinizing hormone-stimulated progesterone production by chicken granulosa cells.     

Roles of Ca2+ and protein kinase C in the excitatory response to serotonin in embryonic molluscan ciliary cells

We examined the roles of Ca2+ and protein kinase C (PKC) in the cilio-excitatory response to serotonin in pedal ciliary cells from Helisoma trivolvis embryos. Serotonin (5-hydroxytryptamine; 5-HT; 100 micromol/L) induced an increase in ciliary beat frequency (CBF) was abolished by microinjected BAPTA (50 mmol/L), but was only partially inhibited by the phospholipase C inhibitor U-73122 (10 micromol/L). The diacylglycerol analogs 1-oleoyl-2-acetyl-sn-glycerol (100 micromol/L) and 1,2-dioctanoyl-sn-glycerol (100 micromol/L) caused increases in [Ca2+]i that were smaller than those induced by serotonin. In the absence of extracellular Ca2+, 1,2-dioctanoyl-sn-glycerol (100 micromol/L) failed to elicit an increase in both CBF and [Ca2+]i. In contrast, the serotonin-induced increase in CBF persisted in the absence of extracellular Ca2+, although the increase in [Ca2+]i was abolished. PKC inhibitors bisindolylmaleimide (10 and 100 nmol/L) and calphostin C (10 nmol/L) partially inhibited the serotonin-induced increase in CBF, but didn't affect the serotonin-induced change in [Ca2+]i. These findings suggest that an intracellular store-dependent increase in [Ca2+]i mediates the cilio-excitatory response to serotonin. Furthermore, although PKC is able to cause an increase in [Ca2+]i through calcium influx, it contributes to the cilio-excitatory response to 5-HT through a different mechanism.     

Heat shock protein synthesis and cytoskeletal rearrangements occur independently of intracellular free calcium increases in Drosophila cells and tissues

Heat shock causes significant changes in intracellular free calcium ([Ca2+]i) which occur rapidly following temperature elevation. The resting level of free calcium in single Drosophila melanogaster larval salivary gland cells measured with the fluorescent indicator fura-2 is 198 +/- 31 nM (n = 4). It increases approximately 10-fold to 1870 +/- 770 nM (n = 4), during a heat shock. When salivary glands are incubated in calcium-free, EGTA-buffered medium the resting free calcium is reduced to 80 +/- 7 nM (n = 3) and heat shock results in a 4-fold increase in free calcium to 353 +/- 90 nM (n = 3). Drosophila Kc cells show a heat shock-induced increase in [Ca2+]i from 118.4 +/- 2 nM (n = 11) to 323 +/- 18 nM. Procedures were devised to block the effects of heat shock on the increase in intracellular calcium and assess its role in the induction of heat shock proteins and in the stress-induced rearrangement of the vimentin cytoskeleton. We report here the changes in [Ca2+]i are not required for a complete induction of the heat shock response or for the collapse of the vimentin cytoskeleton.     

Stimulation of tracheal ciliary beat frequency by capsaicin

To determine the possible involvement of neural and cyclooxygenase pathways whereby irritants might affect cilia activity in vivo, the temporal response of canine tracheal ciliary beat frequency (CBF) to the inhaled surrogate irritant capsaicin was studied. CBF was measured on the ventral midtracheal surface of barbiturate-anesthetized eucapnically ventilated beagle dogs by heterodyne-mode laser light scattering. After base-line CBF was established, hexamethonium bromide (2 mg/kg iv), ipratropium bromide (0.5 microgram/kg iv), indomethacin (2 mg/kg iv), or intravenous 0.9% saline was administered. Aerosolized 3 Z 10(-9) M capsaicin in 0.9% saline was delivered for 2 min, and CBF was measured for the following 60 min. Control experiments used 0.9% saline sham aerosol with a 0.9% saline sham block. Aerosolized capsaicin stimulated CBF from a base line of 6.2 +/- 1.4 (SD) Hz (n = 230) to a mean maximum of 17.7 +/- 7.3 Hz (n = 16) 23 min after aerosol delivery, and CBF returned to base line within 60 min. Neither hexamethonium bromide, ipratropium bromide, nor indomethacin changed CBF from base-line values. The episodic CBF stimulatory response to capsaicin after commencement of aerosol was completely inhibited by hexamethonium bromide. Ipratropium bromide partially inhibited the first 15 min and totally inhibited the following 45 min of stimulatory response. Indomethacin inhibited the initial 15 min but had less effect on the following 45 min of stimulatory response. These data indicate that multiple stimulatory mechanisms function over a prolonged period of time to affect the removal of irritants from the airways and that these mechanisms differ from those involved in the maintenance of basal CBF.     

Effects of cyclopiazonic acid on cytosolic calcium in bovine airway smooth muscle cells

In many cells, inhibition of sarcoplasmic reticulum (SR) Ca2+-ATPase activity induces a steady-state increase in cytosolic calcium concentration ([Ca2+]i) that is sustained by calcium influx. The goal was to characterize the response to inhibition of SR Ca2+-ATPase activity in bovine airway smooth muscle cells. Cells were dispersed from bovine trachealis and loaded with fura 2-AM (0.5 microM) for imaging of single cells. Cyclopiazonic acid (CPA; 5 microM) inhibited refilling of both caffeine- and carbachol-sensitive calcium stores. In the presence of extracellular calcium, CPA caused a transient increase in [Ca2+]i from 166 +/- 11 to 671 +/- 100 nM, and then [Ca2+]i decreased to a sustained level (CPA plateau; 236 +/- 19 nM) significantly above basal. The CPA plateau spontaneously declined toward basal levels after 10 min and was attenuated by discharging intracellular calcium stores. When CPA was applied during sustained stimulation with caffeine or carbachol, decreases in [Ca2+]i were observed. We concluded that the CPA plateau depended on the presence of SR calcium and that SR Ca2+-ATPase activity contributed to sustained increases in [Ca2+]i during stimulation with caffeine and, to a lesser extent, carbachol.     

Cerebral blood flow velocity and cranial fluid volume decrease during +Gz acceleration

Cerebral blood flow (CBF) velocity and cranial fluid volume, which is defined as the total volume of intra- and extracranial fluid, were measured using transcranial Doppler ultrasonography and rheoencephalography, respectively, in humans during graded increase of +Gz acceleration (onset rate: 0.1 G/s) without straining maneuvers. Gz acceleration was terminated when subjects' vision decreased to an angle of less than or equal to 60 degrees, which was defined as the physiological end point. In five subjects, mean CBF velocity decreased 48% from a baseline value of 59.4 +/- 11.2 cm/s to 31.0 +/- 5.6 cm/s (p<0.01) with initial loss of peripheral vision at 5.7 +/- 0.9 Gz. On the other hand, systolic CBF velocity did not change significantly during increasing +Gz acceleration. Cranial impedance, which is proportional to loss of cranial fluid volume, increased by 2.0 +/- 0.8% above the baseline value at the physiological end point (p<0.05). Both the decrease of CBF velocity and the increase of cranial impedance correlated significantly with Gz. These results suggest that +Gz acceleration without straining maneuvers decreases CBF velocity to half normal and probably causes a caudal fluid shift from both intra- and extracranial tissues.     

Hormonal regulation of free intracellular calcium concentrations in small and large ovine luteal cells

The second messengers mediating hormonal regulation of the corpus luteum are incompletely defined, particularly for the primary luteolytic hormone prostaglandin F2 alpha (PGF2 alpha). In this study, hormonally induced changes in free intracellular calcium concentrations were measured in individual small and large ovine luteal cells by using computer-assisted microscopic imaging of fura-2 fluorescence. This technique could readily detect transient increases in free calcium concentrations within both small and large luteal cells after treatment with 1 microM of the calcium ionophore, A23187. Treatment with PGF2 alpha (1 microM) caused a dramatic increase in free calcium concentrations in large (before = 73 +/- 2 nM; 2 min after PGF2 alpha = 370 +/- 21 nM; n = 33 cells) but not in small (before = 66 +/- 4 nM; 2 min after PGF2 alpha = 69 +/- 8 nM; n = 12 cells) luteal cells. The magnitude and timing of the calcium response was dose- and time-dependent. The PGF2 alpha-induced increase in free intracellular calcium is probably due to influx of extracellular calcium, since inclusion of inorganic calcium channel blockers (100 microM manganese or cobalt) attenuated the response to PGF2 alpha and removal of extracellular calcium eliminated the response. In contrast to PGF2 alpha, luteinizing hormone (LH) (100 ng/ml) caused no change in intracellular levels of free calcium in small or large luteal cells, even though this dose of LH stimulated (p less than 0.01) progesterone production by small luteal cells. Therefore, alterations in free calcium concentrations could be the intracellular second message mediating the luteolytic action of PGF2 alpha in the large ovine luteal cell.     

The role of calcium in follicle-stimulating hormone signal transduction in Sertoli cells.

Sertoli cells are hormonally regulated by follicle-stimulating hormone (FSH) acting upon a G-protein-linked cell surface FSH receptor. FSH increases intracellular cyclic AMP but the involvement of other signal transduction mechanisms including intracellular calcium in FSH action are not proven. Using freshly isolated rat Sertoli cells we measured cytosolic free ionized calcium levels by dual-wavelength fluorescence spectrophotometry using the calcium-sensitive fluorescent dye Fura2-AM. The cytosolic calcium concentration in unstimulated Sertoli cells was 89 +/- 2 nM (n = 151 experiments) and was markedly increased by either calcium channel ionophores (ionomycin, Bay K8644) or plasma membrane depolarization consistent with the presence of voltage-sensitive and -independent calcium channel in Sertoli cell membranes. Ovine FSH stimulated a specific, sensitive (ED50, 5.0 ng of S-16/ml), and dose-dependent (maximal at 20 ng/ml) rise in cytosolic calcium commencing within 60 s to reach levels of 192 +/- 31 nM after 180 s and lasting for at least 10 min. The effect of FSH was replicated by forskolin, cholera toxin, and dibutyryl cyclic AMP, suggesting that cyclic AMP may mediate the FSH-induced rise in cytosolic calcium. The FSH-induced rise in cytosolic calcium required extracellular calcium and was abolished by calcium channel blockers specific for dihydropyridine (verapamil, nicardipine), nonvoltage-gated (ruthenium red) or all calcium channels (cobalt). Thus FSH action on Sertoli cells involves a specific, rapid, and sustained increase in cytosolic calcium which requires extracellular calcium and involves both dihydropyridine-sensitive, voltage-gated calcium channels and voltage-independent, receptor-gated calcium channels in the plasma membranes of rat Sertoli cells. The replication by cyclic AMP of the effects of FSH suggests that calcium may be a signal-amplification or -modulating mechanism rather than an alternate primary signal transduction system for FSH in Sertoli cells.     

Soluble adenylyl cyclase is localized to cilia and contributes to ciliary beat frequency regulation via production of cAMP

Ciliated airway epithelial cells are subject to sustained changes in intracellular CO(2)/HCO(3)(-) during exacerbations of airway diseases, but the role of CO(2)/HCO(3)(-)-sensitive soluble adenylyl cyclase (sAC) in ciliary beat regulation is unknown. We now show not only sAC expression in human airway epithelia (by RT-PCR, Western blotting, and immunofluorescence) but also its specific localization to the axoneme (Western blotting and immunofluorescence). Real time estimations of [cAMP] changes in ciliated cells, using FRET between fluorescently tagged PKA subunits (expressed under the foxj1 promoter solely in ciliated cells), revealed CO(2)/HCO(3)(-)-mediated cAMP production. This cAMP production was specifically blocked by sAC inhibitors but not by transmembrane adenylyl cyclase (tmAC) inhibitors. In addition, this cAMP production stimulated ciliary beat frequency (CBF) independently of intracellular pH because PKA and sAC inhibitors were uniquely able to block CO(2)/HCO(3)(-)-mediated changes in CBF (while tmAC inhibitors had no effect). Thus, sAC is localized to motile airway cilia and it contributes to the regulation of human airway CBF. In addition, CO(2)/HCO(3)(-) increases indeed reversibly stimulate intracellular cAMP production by sAC in intact cells.