L-type Ca(2+) channel regulation by pituitary adenylate cyclase-activating polypeptide in vascular myocytes from spontaneously hypertensive rats.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a vasoactive peptide, modulates the L-type Ca(2+) channel current (L channel current) in vascular smooth muscle cells (VSMC) through activation and integration of two intracellular pathways, protein kinase A and protein kinase C (PKC). In the present study we compared the effects of PACAP on the L channel current in VSMC from the spontaneously hypertensive rats (SHR) and normotensive controls, Wistar Kyoto rats (WKY). We found that compared with WKY, VSMC from SHR had a higher L channel current density. Stimulation by PACAP (10 nM) caused an increase in the amplitude of the whole cell current and prolonged open time in VSMC from SHR and WKY, with the increase greater in SHR. These effects of PACAP on the L channel current was mimicked by an activator of PKC. In contrast, PACAP caused a smaller increase in cAMP accumulation in VSMC from SHR than WKY, and there was no difference in the inhibitory effect of 8-bromo-cAMP on the L channel current from both type of cells. The greater increase in amplitude of the L channel current by PACAP in VSMC from SHR persisted in the presence of adenosine cyclic 3',5'-monophosphothioate, Rp-isomer, a cAMP antagonist, but not calphostin C, a PKC inhibitor. Taken together, our results show an increase in L channel current density and an enhanced PACAP effect on the L channel current in VSMC from SHR compared with WKY. This difference in PACAP response appears to be predominately secondary to an increased PKC sensitivity.     

Metabotropic Ca(2+) channel-induced Ca(2+) release and ATP-dependent facilitation of arterial myocyte contraction

Voltage-gated Ca(2+) channels in arterial myocytes can mediate Ca(2+) release from the sarcoplasmic reticulum and, thus, induce contraction without the need of extracellular Ca(2+) influx. This metabotropic action of Ca(2+) channels (denoted as calcium-channel-induced calcium release or CCICR) involves activation of G proteins and the phospholipase C-inositol 1,4,5-trisphosphate pathway. Here, we show a form of vascular tone regulation by extracellular ATP that depends on the modulation of CCICR. In isolated arterial myocytes, ATP produced facilitation of Ca(2+)-channel activation and, subsequently, a strong potentiation of CCICR. The facilitation of L-type channel still occurred after full blockade of purinergic receptors and inhibition of G proteins with GDPbetaS, thus suggesting that ATP directly interacts with Ca(2+) channels. The effects of ATP appear to be highly selective, because they were not mimicked by other nucleotides (ADP or UTP) or vasoactive agents, such as norepinephrine, acetylcholine, or endothelin-1. We have also shown that CCICR can trigger arterial cerebral vasoconstriction in the absence of extracellular calcium and that this phenomenon is greatly facilitated by extracellular ATP. Although, at low concentrations, ATP does not induce arterial contraction per se, this agent markedly potentiates contractility of partially depolarized or primed arteries. Hence, the metabotropic action of L-type Ca(2+) channels could have a high impact on vascular pathophysiology, because, even in the absence of Ca(2+) channel opening, it might mediate elevations of cytosolic Ca(2+) and contraction in partially depolarized vascular smooth muscle cells exposed to small concentrations of agonists.     

Kisspeptin-1 directly stimulates LH and GH secretion from goldfish pituitary cells in a Ca(2+)-dependent manner

It has been established that kisspeptin regulates reproduction via stimulation of hypothalamic gonadotropin-releasing hormone (GnRH) secretion, which then induces pituitary luteinizing hormone (LH) release. Kisspeptin also directly stimulates pituitary hormone release in some mammals. However, in goldfish, whether kisspeptin directly affects pituitary hormone release is controversial. In this study, synthetic goldfish kisspeptin-1((1-10)) (gKiss1) enhances LH and growth hormone (GH) release from primary cultures of goldfish pituitary cells in column perifusion. gKiss1 stimulation of LH and GH secretion were still manifested in the presence of the two native goldfish GnRHs, salmon (s)GnRH (goldfish GnRH-3) and chicken (c)GnRH-II (goldfish GnRH-2), but were attenuated by two voltage-sensitive calcium channel blockers, verapamil and nifedipine. gKiss-induced increases in intracellular Ca(2+) in Fura-2AM pre-loaded goldfish pars distalis cells were also inhibited by nifedipine. These results indicate that, in goldfish, (1) direct gKiss1 actions on pituitary LH and GH secretion exist, (2) these actions are independent of GnRH and (3) they involve Ca(2+) signalling.     

Involvement of protein kinase C and intracellular Ca in goldfish brain somatostatin-28 inhibitory action on growth hormone release in goldfish

Goldfish brain somatostatin-28 (gbSS-28) is present in brain and pituitary tissues of goldfish. We assessed whether gbSS-28 targets Ca²⁺ and/or protein kinase C (PKC)-dependent signaling cascades in inhibiting growth hormone (GH) release. gbSS-28 decreased basal GH release from primary cultures of dispersed goldfish pituitary cells and intracellular free calcium levels ([Ca²⁺]_i) in goldfish somatotropes. gbSS-28 partially reduced [Ca²⁺]_i and GH responses induced by two endogeneous gonadotropin-releasing hormones (GnRHs), salmon (s)GnRH and chicken (c)GnRH-II. Furthermore, gbSS-28 reduced GH increases and abolished [Ca²⁺]_i elevations elicited by two PKC activators, tetradecanoyl 4β-phorbol-13-acetate and dioctanyl glycerol. The PKC inhibitors Gö6976 and Bis II abolished [Ca²⁺]_i responses to PKC activators, but only attenuated GnRH-induced increases in [Ca²⁺]_i and did not alter basal [Ca²⁺]_i. In cells pretreated with Bis II, gbSS-28 further reduced basal [Ca²⁺]_i. Our results suggest that gbSS-28 inhibits GnRH-induced GH release in part by attenuating PKC-mediated GnRH [Ca²⁺]_i signals. gbSS-28 reduces basal GH release also via reduction in [Ca²⁺]_i but PKC is not involved in this regard.     

Novel, thapsigargin-insensitive intracellular Ca(2+) stores control growth hormone release from goldfish pituitary cells

The relative contribution of intracellular Ca(2+) stores to basal and agonist-stimulated hormone release in pituitary cells is still not well understood, especially in non-mammalian vertebrates. Using ratiometric Ca(2+) imaging of single identified goldfish somatotropes, along with time-resolved measurements of growth hormone (GH) secretion, we investigated the Ca(2+)-dependent signal transduction of two endogenous regulators of GH release from the goldfish pituitary. Two gonadotropin-releasing hormones (sGnRH and cGnRH-II) initiated GH release in nominally Ca(2+) free conditions. GnRH-evoked GH release was additive to KCl-stimulated GH responses. Ca(2+) signals and GH release elicited by both GnRHs were abolished by pretreatment with TMB-8, which blocks the release of Ca(2+) from intracellular stores. GnRH-stimulated GH secretion is mediated by caffeine-sensitive intracellular Ca(2+) stores that are functionally independent from those sensitive to thapsigargin and other inhibitors of SERCA-type Ca(2+)/ATPases. The caffeine/TMB-8-sensitive Ca(2+) stores are also involved in spontaneous Ca(2+) signalling and the maintenance of prolonged GH release.     

Involvement of the cAMP messenger system and extracellular Ca(2+) during hyposmotically-induced prolactin release in the Mozambique tilapia

In accord with its role in freshwater osmoregulation, prolactin (PRL) release from the tilapia pituitary is stimulated by small, physiologically relevant reductions in plasma osmolality, a response that is mediated by an acute influx of intracellular Ca²⁺ through stretch-activated Ca²⁺channels. In the present study, the role of the calcium and cyclic AMP (cAMP) messenger system in the transduction of a response to a hyposmotic stimulus was examined using dispersed PRL cells and PRL cell membrane preparations from freshwater-acclimated tilapia. When PRL cells were treated with the phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX) (100 μM), significant increases in cAMP levels and PRL release were observed at 1 h. Exposure to reduced medium osmolality (300 mOsmolal) in the presence of IBMX further augmented PRL release. Depletion of Ca²⁺ from the incubation medium blocked PRL release even in the presence of IBMX. By contrast, exposure of PRL cells to cholera toxin (CTX), an activator of adenylyl cyclase (AC), stimulated PRL release and cAMP accumulation in both the presence and absence of extracellular Ca²⁺. On the other hand, treatment with the Ca²⁺ ionophore A23187, which elicits a large rise in intracellular free Ca²⁺, reduced cAMP accumulation. Likewise, the AC activity of a PRL cell membrane preparation was reduced as extracellular Ca²⁺ concentration increased from 0.1 to 1 μM. These results indicate that: (1) the stimulation of PRL release and cAMP formation by a fall in extracellular osmolality are Ca²⁺-dependent; (2) large increases in intracellular Ca²⁺ attenuate cAMP formation; (3) direct agonists of cAMP messenger system, such as cholera toxin, however, stimulate PRL release independently of the extracellular Ca²⁺. These findings add to the evidence that the osmosensitive response of the tilapia PRL cell is mediated through a Ca²⁺-dependent mechanism. Nevertheless, the present findings also suggest that tilapia PRL cells have the ability to rapidly augment release PRL both via a Ca²⁺-dependent manner and via a cAMP-dependent pathway in the absence of extracellular Ca²⁺.     

Dependence of prolactin release on coupling between Ca(2+) mobilization and voltage-gated Ca(2+) influx pathways in rat lactotrophs

Two Ca²⁺-mobilizing receptors expressed in lactotrophs, endothelin-A (ET_A) and thyrotropin-releasing hormone (TRH), induce a rapid Ca²⁺ release from intracellular stores and prolactin (PRL) secretion but differ in their actions during the sustained stimulation; TRH facilitates and ET-1 inhibits voltage-gated calcium influx (VGCI) and PRL secretion. In pertussis toxin (PTX)-treated cells, ET-1-induced inhibition of VGCI was abolished and the pattern of Ca²⁺ signaling was highly comparable with that observed in TRH-stimulated cells. The addition of Cs⁺, a relatively specific blocker of inward rectifier K⁺ channels, mimicked the effect of PTX on the pattern of ET-1-induced sustained Ca²⁺ signaling, but only in about 50% of cells, and did not affect agonist-induced inhibition of PRL secretion. Extracellular Cs⁺ was also ineffective in altering the TRH-induced facilitation of VGCI and PRL secretion. Furthermore, apamin and paxilline, specific blockers of Ca²⁺-activated SK-and BK-type K⁺ channels, respectively; E-4031, a blocker of ether a-go-go K⁺ channel; and linopirdine, a blocker of M-type K⁺ channel, did not affect the agonist-specific patterns of calcium signaling and PRL secretion. These results suggest that ET-1 inhibits VGCI through activation of Cs⁺-sensitive channels, presumably the G_i/o-controlled inward rectifier K⁺ channels, and that this agonist also inhibits PRL release, but downstream of Ca²⁺ influx. Further studies are required to identify the mechanism of sustained TRH-induced facilitation of VGCI and PRL secretion.     

Pituitary adenylate cyclase activating polypeptide, growth hormone (GH)-releasing peptide and GH-releasing hormone stimulate GH release through distinct pituitary receptors.

GH secretion has been thought traditionally to be regulated by the two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin (SRIF). Recent evidence has suggested that other factors may be involved. These factors include the natural ligand for the synthetic hexapeptide GH-releasing peptide (GHRP) and the putative hypophysiotropic factor pituitary adenylate cyclase-activating polypeptide (PA-CAP). Accordingly, we examined the effects of GHRP and PACAP on GH secretion at the single cell level using the reverse hemolytic plaque assay which allows distinction of effects on the number of secreting cells and the amount of hormone each cell secretes. Both factors stimulated GH secretion in a dose-dependent fashion, with PACAP being more effective. PACAP increased both the number of cells secreting and the mean amount of hormone secreted per cell. In contrast, GHRP increased the number of secreting cells, although it had no effect on the amount of secretion per cell. GH secretion induced by GHRH, GHRP, and PACAP was inhibited by SRIF, but the effect was predominantly on the number of cells secreting rather than the amount secreted per cell. Specific antagonists to GHRP and GHRH inhibited GH secretion induced by the respective agonist but not that induced by the other factor nor by PACAP. These findings confirm the complex nature of the regulation of GH secretion at the level of the somatotrope. At least three factors, operating via distinct receptors, are able to increase GH secretion. In addition, they ascribe a potential physiological role for the hitherto putative hypophysiotropic factor PACAP.     

The effects of maitotoxin on 45Ca2+ flux and hormone release in GH3 rat pituitary cells

Maitotoxin has been reported to activate calcium channels and stimulate calcium-dependent functions in several tissues, but a thorough investigation of 45Ca2+ fluxes is lacking. To characterize the influence of maitotoxin on 45Ca2+ flux in greater detail, we incubated dispersed GH3 pituitary tumor cells in 45Ca2+ with maitotoxin and other agents affecting calcium channels. Within 10 sec of exposure, maitotoxin induced a net calcium influx in cells at isotopic equilibrium. Calcium uptake was concentration dependent between 0.4 and 40 ng/ml maitotoxin and was inhibited by antagonists of voltage-dependent calcium channels but not by inhibitors of sodium channels. PRL and GH release from perifused GH3 cells was stimulated within 1 min by maitotoxin. We conclude that maitotoxin causes a rapid, concentration-dependent influx of calcium through presumed voltage-dependent endogenous calcium channels, culminating in enhanced hormone release. This potent toxin may provide a more precise understanding of the role of calcium in the stimulus-secretion coupling process.     

Nitric oxide and hydroperoxide affect islet hormone release and Ca(2+) efflux

We have investigated the influence of the intracellular free radical donors hydroxylamine (giving nitric oxide [NO]) and tert-butylhydroperoxide (giving hydroperoxide [“H₂O₂”]) on glucose- and cyclic adenosine monophosphate (cAMP)-induced transduction signaling in islet hormone release. Both donors dose dependently inhibited glucose-stimulated insulin release and induced modest (hydroxylamine) or profound (tert-butylhydroperoxide) suppression of ⁴⁵Ca²⁺-efflux from perifused islets. By contrast, both donors stimulated glucagon release. Similar effects on hormone release were displayed after K⁺-depolarization. Insulin and glucagon release stimulated by activation of the cAMP system through isobutylmethylxanthine (IBMX) at basal glucose was modestly potentiated by low concentrations of both donors. These effects were still observed, although less pronounced, in K⁺-depolarized islets. In vitro as well as in vivo, the NO-synthase inhibitor N^G-nitro-L-arginine methyl ester inhibited IBMX-induced glucagon release, but did not affect insulin release. The results suggest that NO and hydroperoxide inhibit glucose-stimulated insulin release by perturbing Ca²⁺ fluxes and probably acting through S-nitrosylation (NO) or oxidation (hydroperoxide) of thiol groups critical to the secretory process. These effects are largely independent of depolarization events. By contrast, both NO and hydroperoxide can potentiate cAMP-stimulated hormone release presumably at a distal site in the stimulus-secretion coupling.     

Cortisol rapidly reduces prolactin release and cAMP and 45Ca2+ accumulation in the cichlid fish pituitary in vitro

During in vitro incubation, prolactin release is inhibited in a dose-related manner by cortisol. This action is mimicked by the synthetic glucocorticoid agonist dexamethasone but not by other steroids tested. Perifusion studies indicate that the inhibition of [3H]prolactin release by cortisol occurs within 20 min. Cortisol (50 nM) also inhibits cAMP accumulation and reduces 45Ca2+ accumulation in the tilapia rostral pars distalis within 15 min. Cortisol's action on prolactin release is blocked in the presence of either the Ca2+ ionophore A23187 or a combination of dibutyryl cAMP and 3-isobutyl-1-methylxanthine, which increase intracellular Ca2+ and cAMP, respectively. Taken together, these findings suggest that cortisol may play a physiologically relevant role in the rapid modulation of prolactin secretion in vivo. Our studies also suggest that the inhibition of prolactin release by cortisol is a specific glucocorticoid action that may be mediated, in part, through cortisol's ability to inhibit intracellular cAMP and Ca2+ metabolism.     

Pituitary adenylate cyclase-activating polypeptide releases 7B2, adrenocorticotrophin, growth hormone and prolactin from the mouse and rat clonal pituitary cell lines AtT-20 and GH3.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from ovine hypothalami and so called because of its ability to stimulate pituitary adenylate cyclase activity. Alternative amidation and proteolytic processing of prepro-PACAP gives rise to two bioactive-amidated forms, PACAP-NH2(1-38) (PACAP-38) and PACAP-NH2(1-27) (PACAP-27). 7B2 is a polypeptide of 185 amino acids which is predominantly found in secretory granules and is widely distributed in rat and human tissues. We investigated the ability of the two forms of PACAP to stimulate GH, prolactin and 7B2 release by the rat pituitary clonal cell line GH3, and ACTH and 7B2 by the mouse pituitary clonal cell line AtT-20. PACAP-38 and PACAP-27 stimulated 7B2 and GH/prolactin or ACTH secretion with a similar efficacy over the 2-h incubation period from GH3 and AtT-20 cells respectively. 7B2 secretion was also stimulated by corticotrophin-releasing factor (CRF-41) and vasoactive intestinal polypeptide (VIP) in AtT-20 cells, and thyrotrophin-releasing hormone (TRH) and VIP in GH3 cells. Addition of PACAP to CRF-41 resulted in an additive effect on ACTH secretion and a synergistic effect on 7B2 secretion in AtT-20 cells. No synergism was observed when PACAP was added together with TRH, either on GH and prolactin secretion or on 7B2 release from GH3 cells. PACAP-mediated 7B2 secretion from both cell lines and PACAP-stimulated ACTH release from AtT-20 cells were reduced by 5 mg octapeptide synthetic somatostatin analogue/l (5 mg SMS 201-995/l).     

Ca(2+)-inhibitable adenylyl cyclase modulates pulmonary artery endothelial cell cAMP content and barrier function.

Maintenance by the endothelium of a semi-permeable barrier is critically important in the exchange of oxygen and carbon dioxide in the lung. Intracellular free Ca2+ ([Ca2+]i) and cAMP are principal determinants of endothelial cell barrier function through their mutually opposing actions on endothelial retraction. However, details of the mechanisms of this antagonism are lacking. The recent discovery that certain adenylyl cyclases (EC 4.6.1.1) could be acutely inhibited by Ca2+ in the intracellular concentration range provided one possible mechanism whereby elevated [Ca2+]i could decrease cAMP content. This possibility was explored in pulmonary artery endothelial cells. The results indicate that a type VI Ca(2+)-inhibitable adenylyl cyclase exists in pulmonary artery endothelial cells and is modulated by physiological changes in [Ca2+]i. Furthermore, the results suggest the inverse relationship between [Ca2+]i and cAMP that is established by Ca(2+)-inhibitable adenylyl cyclase plays a critical role in modulating pulmonary artery endothelial cell permeability. These data provide evidence that susceptibility to inhibition of adenylyl cyclase by Ca2+ can be exploited in modulating a central physiological process.     

Coordinated control of cell Ca(2+) loading and triggered release from the sarcoplasmic reticulum underlies the rapid inotropic response to increased L-type Ca(2+) current

The aim of this study was to investigate how sarcoplasmic reticulum (SR) Ca(2+) content and systolic Ca(2+) are controlled when Ca(2+) entry into the cell is varied. Experiments were performed on voltage-clamped rat and ferret ventricular myocytes loaded with fluo-3 to measure intracellular Ca(2+) concentration ([Ca(2+)](i)). Increasing external Ca(2+) concentration ([Ca(2+)](o)) from 1 to 2 mmol/L increased the amplitude of the systolic Ca(2+) transient with no effect on SR Ca(2+) content. This constancy of SR content is shown to result because the larger Ca(2+) transient activates a larger Ca(2+) efflux from the cell that balances the increased influx. Decreasing [Ca(2+)](o) to 0.2 mmol/L decreased systolic Ca(2+) but produced a small increase of SR Ca(2+) content. This increase of SR Ca(2+) content is due to a decreased release of Ca(2+) from the SR resulting in decreased loss of Ca(2+) from the cell. An increase of [Ca(2+)](o) has two effects: (1) increasing the fraction of SR Ca(2+) content, which is released on depolarization and (2) increasing Ca(2+) entry into the cell. The results of this study show that the combination of these effects results in rapid changes in the amplitude of the systolic Ca(2+) transient. In support of this, the changes of amplitude of the transient occur more quickly following changes of [Ca(2+)](o) than following refilling of the SR after depletion with caffeine. We conclude that the coordinated control of increased Ca(2+) entry and greater fractional release of Ca(2+) is an important factor in regulating excitation-contraction coupling.     

Spatiotemporal characteristics of SR Ca(2+) uptake and release in detubulated rat ventricular myocytes

In cardiac ventricular myocytes, sarcoplasmic reticulum (SR) Ca(2+) load is a key determinant of SR Ca(2+) release. This release normally occurs predominantly from SR junctions at sarcolemmal invaginations (t-tubules), ensuring synchronous SR Ca(2+) release throughout the cell. However under conditions of Ca(2+) overload, spontaneous SR Ca(2+) release and propagating Ca(2+) waves can occur, which are pro-arrhythmic. We used detubulated rat ventricular myocytes to determine the dependence of Ca(2+) wave propagation on SR Ca(2+) load, and the role of t-tubules in SR Ca(2+) uptake and spontaneous release. After SR Ca(2+) depletion, recovery of Ca(2+) transient amplitude (and SR Ca(2+) load) was slower in detubulated than control myocytes (half-maximal recovery: 9.9+/-1.4 vs. 5.5+/-0.7 beats). In detubulated myocytes the extent and velocity of Ca(2+) propagation from the cell periphery increased with each beat and depended steeply on SR Ca(2+) load. Isoproterenol (ISO) accelerated recovery, increased maximal propagation velocity and reduced the threshold SR Ca(2+) load for propagation. Ca(2+) spark frequency was uniform across control cell width and was similar at the periphery of detubulated cells. However, internal Ca(2+) spark frequency in detubulated cells was 75% lower (despite comparable local SR Ca(2+) load); this transverse spark frequency profile was similar to that in atrial myocytes. We conclude that: (1) t-tubule Ca(2+) fluxes normally control SR Ca(2+) refilling; (2) Ca(2+) wave propagation depends steeply on SR Ca(2+) content (3) SR-t-tubule junctions are important in initiating SR Ca(2+) release and (4) ISO enhances propagation of SR Ca release, but not the initiation of SR Ca release events (for given SR Ca(2+) loads).     

Association of 45Ca2+ mobilization with stimulation of growth hormone (GH) release by GH-releasing factor in dispersed normal male rat pituitary cells

Dispersed normal male rat anterior pituitary cells were prelabeled with 45Ca2+ and perifused to study the influence of GH-releasing factor (GRF) on fractional calcium efflux and GH release. The cells were exposed for 2 min to 0, 0.03, 0.1, 0.3, 1.0, or 10.0 nM GRF in separate perifusion columns, and the response to each concentration was determined by integration of the area under the curve. Concentrations of 0.1 nM GRF and higher produced a simultaneous and significant stimulation of calcium efflux and GH release. The increase in calcium efflux was proportional to GRF concentration and was maximally responsive at 1 nM GRF. The value for the entire integrated response of GH release increased continuously with GRF concentration, but GH released rapidly (0-4 min) in response to GRF achieved a maximal response at 1 nM GRF and was significantly correlated with calcium efflux. Somatostatin (100 nM) abolished the stimulation of GH release and calcium efflux due to 10 nM GRF. We conclude that GRF receptor activation is intimately associated with calcium mobilization, although the relative dependence upon intracellular or extracellular calcium sources has yet to be defined. This interaction occurs at a GRF concentration about 10 times lower than that observed to cause a measurable increase in intracellular biochemical messengers such as cAMP, phosphatidylinositol, or arachidonate. We postulate that GRF-stimulated calcium mobilization is a rapid and very sensitive event contributing to GRF-stimulated GH release.     

Alpha-1 adrenergic receptor agonists modulate ductal secretion of BDL rats via Ca(2+)- and PKC-dependent stimulation of cAMP

Acetylcholine potentiates secretin-stimulated ductal secretion by Ca(2+)-calcineurin-mediated modulation of adenylyl cyclase. D2 dopaminergic receptor agonists inhibit secretin-stimulated ductal secretion via activation of protein kinase C (PKC)-gamma. No information exists regarding the effect of adrenergic receptor agonists on ductal secretion in a model of cholestasis induced by bile duct ligation (BDL). We evaluated the expression of alpha-1A/1C, -1beta and beta-1 adrenergic receptors in liver sections and cholangiocytes from normal and BDL rats. We evaluated the effects of the alpha-1 and beta-1 adrenergic receptor agonists (phenylephrine and dobutamine, respectively) on bile and bicarbonate secretion and cholangiocyte IP(3) and Ca(2+) levels in normal and BDL rats. We measured the effect of phenylephrine on lumen expansion in intrahepatic bile duct units (IBDUs) and cyclic adenosine monophosphate (cAMP) levels in cholangiocytes from BDL rats in the absence or presence of BAPTA/AM and G?6976 (a PKC-alpha inhibitor). We evaluated if the effects of phenylephrine on ductal secretion were associated with translocation of PKC isoforms leading to increased protein kinase A activity. Alpha-1 and beta-1 adrenergic receptors were present mostly in the basolateral domain of cholangiocytes and, following BDL, their expression increased. Phenylephrine, but not dobutamine, increased secretin-stimulated choleresis in BDL rats. Phenylephrine did not alter basal but increased secretin-stimulated IBDU lumen expansion and cAMP levels, which were blocked by BAPTA/AM and Go6976. Phenylephrine increased IP(3) and Ca(2+) levels and activated PKC-alpha and PKC-beta-II. In conclusion, coordinated regulation of ductal secretion by secretin (through cAMP) and adrenergic receptor agonist activation (through Ca(2+)/PKC) induces maximal ductal bicarbonate secretion in liver diseases. (Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).