VIP and muscarinic synergistic mucin secretion by salivary mucous cells is mediated by enhanced PKC activity via VIP-induced release of an intracellular Ca2+ pool.

Mucin secretion by salivary mucous glands is mediated predominantly by parasympathetic acetylcholine activation of cholinergic muscarinic receptors via increased intracellular free calcium ([Ca2+]i) and activation of conventional protein kinase C isozymes (cPKC). However, the parasympathetic co-neurotransmitter, vasoactive intestinal peptide (VIP), also initiates secretion, but to a lesser extent. In the present study, cross talk between VIP- and muscarinic-induced mucin secretion was investigated using isolated rat sublingual tubuloacini. VIP-induced secretion is mediated by cAMP-activated protein kinase A (PKA), independently of increased [Ca2+]i. Synergistic secretion between VIP and the muscarinic agonist, carbachol, was demonstrated but only with submaximal carbachol. Carbachol has no effect on cAMP ± VIP. Instead, PKA activated by VIP releases Ca2+ from an intracellular pool maintained by the sarco/endoplasmic reticulum Ca2+-ATPase pump. Calcium release was independent of phospholipase C activity. The resultant sustained [Ca2+]i increase is additive to submaximal, but not maximal carbachol-induced [Ca2+]i. Synergistic mucin secretion was mimicked by VIP plus either phorbol 12-myristate 13-acetate or 0.01 µM thapsigargin, and blocked by the PKC inhibitor, Gö6976. VIP-induced Ca2+ release also promoted store-operated Ca2+ entry. Synergism is therefore driven by VIP-mediated [Ca2+]i augmenting cPKC activity to enhance muscarinic mucin secretion. Additional data suggest ryanodine receptors control VIP/PKA-mediated Ca2+ release from a Ca2+ pool also responsive to maximal carbachol. A working model of muscarinic and VIP control of mucous cell exocrine secretion is presented. Results are discussed in relation to synergistic mechanisms in other secretory cells, and the physiological and therapeutic significance of VIP/muscarinic synergism controlling salivary mucous cell exocrine secretion.

Sorafenib induces apoptosis of AML cells via Bim-mediated activation of the intrinsic apoptotic pathway.

Raf/MEK/Erk signaling is activated in the majority of acute myeloid leukemias (AMLs), providing rationale for targeting this pathway with therapeutic intent. We investigated growth-inhibitory and proapoptotic effects of sorafenib in AML. Our studies demonstrated that sorafenib significantly inhibited the phosphorylation levels of Raf downstream target proteins MEK1/2 and Erk, induced apoptosis and inhibited colony formation in AML cell lines and in primary AML samples. Mechanistically, treatment with sorafenib resulted in upregulation of proapoptotic Bim, accompanied by an increase in Bad, Bax and Bak protein levels and decreased Mcl-1, X-linked inhibitor of apoptosis and surviving levels, which mainly led to the activation of the intrinsic apoptotic pathway. Silencing of Bim protein expression significantly abrogated sorafenib-induced apoptosis, suggesting a critical function of Bim in the activation of the intrinsic mitochondrial pathway induced by sorafenib. Importantly, sorafenib also modulated phospho-Erk, Bim, Bax and Mcl-1 levels in samples procured from patients in an ongoing Phase I clinical trial of sorafenib in AML. Combination of sorafenib with cytarabine or the novel small molecule Bcl-2 inhibitor ABT-737 synergistically induced cell death in AML cell lines. Our results strongly suggest potential activity of sorafenib as a novel mechanism-based therapeutic agent in AML.

Survivorship of the St Georg Sled medial unicompartmental knee replacement beyond ten years.

There have been several reports of good survivorship and excellent function at ten years with fixed-bearing unicompartmental knee replacement. However, little is known about survival beyond ten years. From the Bristol database of over 4000 knee replacements, we identified 203 St Georg Sled unicompartmental knee replacements (174 patients) which had already survived ten years. The mean age of the patients at surgery was 67.1 years (35.7 to 85) with 67 (38.5%) being under 65 years at the time of surgery. They were reviewed at a mean of 14.8 years (10 to 29.4) from surgery to determine survivorship and function. There were 99 knees followed up for 15 years, 21 for 20 years and four for 25 years. The remainder failed, were withdrawn, or the patient had died. In 58 patients (69 knees) the implant was in situ at the time of death. Revision was undertaken in 16 knees (7.9%) at a mean of 13 years (10.2 to 21.6) after operation. In seven knees (3.4%) this was for progression of arthritis, in three (1.5%) for wear of polyethylene, in four (2%) for tibial loosening, in two (1%) for fracture of the femoral component and in two (1%) for infection. Two knees (1%) were revised for more than one reason. The mean Bristol knee score of the surviving knees fell from 86 (34 to 100) to 79 (42 to 100) during the second decade. Survivorship to 20 years was 85.9% (95% CI 82.9% to 88.9%) and at 25 years was 80% (95% CI 70.2% to 89.8%). Satisfactory survival of a fixed-bearing unicompartmental knee replacement can be achieved into the second decade and beyond.

Hidden blood loss following hip and knee arthroplasty. Correct management of blood loss should take hidden loss into account.

Following total hip arthroplasty (THA) and total knee arthroplasty (TKR) only the 'visible' measured blood loss is usually known. This underestimates the 'true' total loss, as some loss is 'hidden'. Correct management of blood loss should take hidden loss into account. We studied 101 THAs and 101 TKAs (with re-infusion of drained blood). Following THA, the mean total loss was 1510 ml and the hidden loss 471 ml (26%). Following TKA, the mean total loss was 1498 ml. The hidden loss was 765 ml (49%). Obesity made no difference with either operation. THA involves a small hidden loss, the total loss being 1.3 times that measured. However, following TKA, there may be substantial hidden blood loss due to bleeding into the tissues and residual blood in the joint. The true total loss can be determined by doubling the measured loss.

Lateral unicompartmental knee replacement survivorship and clinical experience over 21 years.

We describe 88 knees (79 patients) with lateral unicompartmental osteoarthritis which had been treated by the St Georg Sled prosthesis. At a mean follow-up of nine years (2 to 21) 15 knees had revision surgery, nine for progression of arthritis, six for loosening, four for breakage of a component and four for more than one reason. Six patients complained of moderate or severe pain at the final follow-up. Only five knees were lost to follow-up in the 21-year period. We performed survivorship analysis on the group using revision for any cause as the endpoint. At ten years the cumulative survival rate was 83%, and at 15 years, when ten knees were still at risk, it was 74%. Based on our clinical results and survival rate the St Georg Sled may be considered to be a suitable unicompartmental replacement for isolated lateral compartment osteoarthritis.

What can transgenic and gene-targeted mouse models teach us about salivary gland physiology?

Thousands of genetically modified mice have been developed since the first reports of stable expression of recombinant DNA in this species nearly 20 years ago. This mammalian model system has revolutionized the study of whole-animal, organ, and cell physiology. Transgenic and gene-targeted mice have been widely used to characterize salivary-gland-specific expression and to identify genes associated with tumorigenesis. Moreover, several of these mouse lines have proved to be useful models of salivary gland disease related to impaired immunology, i.e., Sjögren's syndrome, and disease states associated with pathogens. Despite the availability of genetically modified mice, few investigators have taken advantage of this resource to better their understanding of salivary gland function as it relates to the production of saliva. In this article, we describe the methods used to generate transgenic and gene-targeted mice and provide an overview of the advantages of and potential difficulties with these models. Finally, using these mouse models, we discuss the advances made in our understanding of the salivary gland secretion process.

Persistence of p53 mutations and resistance of keratinocytes to apoptosis are associated with the increased susceptibility of mice lacking the XPC gene to UV carcinogenesis.

Like xeroderma pigmentosum (XP) patients, transgenic mice lacking nucleotide excision repair (NER) genes such as XPA and XPC are extremely susceptible to ultraviolet (UV)-induced skin cancer. Because the p53 gene is an important target for UV carcinogenesis and because the p53 protein modulates NER, we investigated the consequences of NER deficiency on UV-induced p53 mutations in XPC-/- mouse skin tumors. Thirty-eight (76%) of 50 UV-induced XPC-/- skin tumor analysed displayed C-->T or CC-->TT transitions at dipyrimidine sites on the untranscribed strand of the p53 gene. A major hot spot for p53 mutation occurred at codon 270, which is also a hot spot in UV-induced skin tumors from NER-proficient C3H and SKH-hr 1 mice. Interestingly, codon 270 mutations were induced in both XPC-/- and +/+ mouse skin after 1 week of UV irradiation, but the mutations persisted only in XPC-/- mouse skin after 3 - 4 weeks of chronic UV. The persistence of UV-induced p53 mutations in XPC-/- mouse skin was associated with decreased apoptosis and increased proliferation of keratinocytes, suggesting that these events may contribute to the accelerated development of UV-induced skin tumors in XPC-/- mice.

Radiation-induced progressive decrease in fluid secretion in rat submandibular glands is related to decreased acinar volume and not impaired calcium signaling.

The mechanism(s) of radiation-induced salivary gland dysfunction is poorly understood. In the present study, we have assessed the secretory function (muscarinic agonist-stimulated saliva flow, intracellular calcium mobilization, Na+/K+/2Cl- cotransport activity) in rat submandibular glands 12 months postirradiation (single dose, 10 Gy). The morphological status of glands from control and irradiated rats was also determined. Pilocarpine-stimulated salivary flow was decreased by 67% at 12 months (but not at 3 months) after irradiation. This was associated with a 47% decrease in the wet weight of the irradiated glands. Histological and morphometric analysis demonstrated that acinar cells were smaller and occupied relatively less volume and convoluted granular tubules were smaller but occupied the same relative volume, while intercalated and striated ducts maintained their size but occupied a greater relative volume in submandibular glands from irradiated compared to control animals. In addition, no inflammation or fibrosis was observed in the irradiated tissues. Carbachol- or thapsigargin-stimulated mobilization of Ca2+ was similar in dispersed submandibular gland cells from control and irradiated animals. Further, [Ca2+]i imaging of individual ducts and acini from control and irradiated groups showed, for the first time, that mobilization of Ca2+ in either cell type was not altered by the radiation treatment. The carbachol-stimulated, bumetanide-sensitive component of the Na+/K+/ 2Cl- cotransport activity was also similar in submandibular gland cells from control and irradiated animals. These data demonstrate that a single dose of gamma radiation induces a progressive loss of submandibular gland tissue and function. This loss of salivary flow is not due to chronic inflammation or fibrosis of the gland or an alteration in the neurotransmitter signaling mechanism in the acinar or ductal cells. The radiation-induced decrease in fluid secretion appears to be related to a change in either the water-handling capacity of the acini or the number of acinar cells in the gland.

In vitro studies of the regulation of mucous gland secretion.

Our laboratory is concerned with elucidation of mechanisms regulating the exocrine secretion of mucin glycoproteins by mucous cells of salivary mucous glands. As a model system for our studies we use short-term cultures of acinar structures isolated from rat sublingual glands. Recent results are discussed from an on-going study of cross-talk between the two primary signaling pathways regulating exocrine secretion from isolated sublingual acini: the muscarinic cholinergic and vasoactive intestinal peptide (VIP) pathways. The combination of muscarinic agonist (carbachol) and VIP elicits a secretion equivalent to about 150% of the additive sum of the secretory responses to each agonist alone. This synergistic secretory response is only observed at submaximal concentrations of carbachol. VIP thus serves to decrease the EC50 for carbachol nearly three-fold. Results are thus far consistent with the hypothesis that a sustained rise in the intracellular concentration of calcium ions induced by VIP accounts for synergistic secretion and the heightened sensitivity to carbachol.

Evidence for a physiological role of NH4+ transport on the secretory Na(+)-K(+)-2Cl- cotransporter.

The secretory Na(+)-K(+)-2Cl- cotransporter in salivary acinar cells is responsible for driving the transepithelial Cl- fluxes that give rise to fluid secretion. We demonstrate that the application of the muscarinic agonist carbachol to rat parotid acini results in an intracellular acid load that can be blocked by bumetanide, a specific inhibitor of the cotransporter. One component of this bumetanide-sensitive acid load is ouabain-sensitive while a second is dependent on the presence of sub-millimolar concentrations of NH4+ in our media. Our data indicate that this latter effect arises from NH4+ entry on the cotransporter operating in a Na(+)-NH4(+)-2Cl- cotransport mode and that at physiological NH4+ levels in the rat (approximately 0.1 mM), 10-15% of the acinar Cl- entry occurs via this route. We suggest that Na(+)-NH4(+)-2Cl- cotransport may also play a significant physiological role in other cell types and that this mode of operation of the secretory Na(+)-K(+)-2Cl- cotransporter could account for the currently unexplained presence of this protein in a number of tissues.

p53 Mutations in hairless SKH-hr1 mouse skin tumors induced by a solar simulator.

In this study, we investigated whether the spectrum of p53 mutations in skin tumors induced in hairless SKH-hr1 mice by a solar simulator (290-400 nm) are similar to those found in skin tumors induced in C3H mice by UV radiation from unfiltered (250-400 nm) and Kodacel-filtered (290-400 nm) FS40 sunlamps. Analysis of tumor DNA for p53 mutations revealed that 14 of 16 (87.5%) SkH-hr1 skin tumors induced by the solar simulator contained mutations. Single C-->T transitions at dipyrimidine sequences located on the nontranscribed DNA strand were the most predominant type of p53 mutation. Remarkably, 52% of all p53 mutations in solar simulator-induced SKH-hr1 skin tumors occurred at codon 270, which is also a hotspot in C3H skin tumors induced by unfiltered and Kodacel-filtered FS40 sunlamps. However, T-->G transversions, which are hallmarks of UVA-induced mutations, were not detected in any of the solar simulator-induced skin tumors analyzed. These results demonstrate that the p53 mutation spectra seen in solar simulator-induced SKH-hr1 skin tumors are similar to those present in -unfiltered and Kodacel-filtered FS40 sunlamp-induced C3H skin tumors. In addition, our data indicate that the UVA present in solar simulator radiation does not play a role in the induction of p53 mutations that contribute to skin cancer development.

Molecular analysis of the human vitamin D binding protein (group specific component, Gc) in tuberous sclerosis complex (TSC).

Group specific component (Gc) is an abundant plasma protein whose functional role is not clearly established. Gc protein is synthesised in the liver and is known to bind vitamin D, vitamin D metabolites, and G actin; Gc protein is also implicated in macrophage activation. Several polymorphic electrophoretic variants of Gc protein are found in all human populations; the most common alleles are Gc-1f, Gc-1s, and Gc-2. In previous studies, Gc allele frequencies, determined using isoelectric focusing or immunofixation or both, were significantly different in patients with tuberous sclerosis complex (TSC) from matched controls, with an excess of Gc-2 in patients. Linkage association between Gc and TSC is unlikely since the Gc locus maps to chromosome 4q12, whereas the two common forms of TSC map to 9q34 and 16p13.1, respectively. However, a direct cause and effect relationship between Gc protein and TSC symptoms is possible. To investigate further the relationship between the Gc locus and TSC, two Gc restriction site polymorphisms, HaeIII and StyI, were typed in 43 unrelated white subjects with TSC. The frequencies of the restriction site polymorphisms in the TSC patients did not differ from those in control populations. Therefore a direct association between Gc type and TSC is unlikely. The previously reported association was either spurious or the result of typing errors in plasma from subjects with underlying abnormalities in plasma proteins.

Sunlight and skin cancer: inhibition of p53 mutations in UV-irradiated mouse skin by sunscreens.

UV-induced mutations in the p53 tumor suppressor gene play an essential role in skin cancer development. We report here that such mutations can be detected in UV-irradiated mouse skin months before the gross appearance of skin tumors. Application of SPF-15 sunscreens to mouse skin before each UV irradiation nearly abolished the frequency of p53 mutations. These results indicate that p53 mutation is an early event in UV skin carcinogenesis and that inhibition of this event may serve as an early end point for assessing protective measures against skin cancer development.

Interactions between secretin and acetylcholine in the regulation of fluid secretion by isolated rat pancreatic ducts.

1. Interlobular ducts were isolated from the rat pancreas and maintained in short-term tissue culture. Fluid secretion from these isolated ducts was measured using micropuncture techniques, intracellular calcium concentration ([Ca2+]i) by fura-2 microspectrofluorimetry, and cyclic AMP by radioimmunoassay. 2. Applying secretin and ACh simultaneously to ducts caused either a stimulation or an inhibition of fluid secretion depending on the doses employed. 3. The inhibitory effect of secretin and ACh could be relieved by atropine, and by the protein kinase C (PKC) inhibitors staurosporine and 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7). 4. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol 12, 13-dibutyrate (PDBu) inhibited secretin-evoked fluid secretion. 5. ACh and TPA also inhibited fluid secretion stimulated by the adenylate cyclase activator, forskolin. 6. Neither secretin nor the PKC activators and inhibitors had any effect on either the increase in [Ca2+]i evoked by ACh or the increase in intracellular cyclic AMP evoked by secretin and forskolin. 7. We conclude that the inhibitory effect of combined doses of secretin and ACh on ductal fluid secretion is probably mediated by PKC at a point in the secretory mechanism distal to the generation of intracellular messengers.

Transfection of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 cells.

Aggregation of the high affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells results in rapid tyrosine phosphorylation and activation of Syk, a cytoplasmic protein tyrosine kinase. To examine the role of Syk in the Fc epsilon RI signaling pathway, we identified a variant of RBL-2H3 cells that has no detectable Syk by immunoblotting and by in vitro kinase reactions. In these Syk-deficient TB1A2 cells, aggregation of Fc epsilon RI induced no histamine release and no detectable increase in total cellular protein tyrosine phosphorylation. However, stimulation of these cells with the calcium ionophore did induce degranulation. Fc epsilon RI aggregation induced tyrosine phosphorylation of the beta and gamma subunits of the receptor, but no increase in the tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2 and no detectable increase in intracellular free Ca2+ concentration. By transfection, cloned lines were established with stable expression of Syk. In these reconstituted cells, Fc epsilon RI aggregation induced tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2, an increase in intracellular free Ca2+ and histamine release. These results demonstrate that Syk plays a critical role in the early Fc epsilon RI-mediated signaling events. It further demonstrates that Syk activation occurs downstream of receptor phosphorylation, but upstream of most of the Fc epsilon RI-mediated protein tyrosine phosphorylations.

Elevation of intracellular cAMP by noradrenaline and vasoactive intestinal peptide in striated ducts isolated from the rabbit mandibular salivary gland.

Salivary gland intralobular ducts are responsible for the modification of the electrolyte composition of the primary fluid secreted by the acini. However, the intracellular messengers that regulate this and other intralobular duct cell processes have not been fully characterized. To investigate the possibility that cAMP-mobilizing agonists may be involved, intralobular (striated) ducts were isolated from the rabbit mandibular salivary gland by tissue dissociation and microdissection and maintained in tissue culture overnight. Individual duct fragments were stimulated with the secretory agonists noradrenaline, vasoactive intestinal peptide (VIP) and substance P and their cAMP content measured by acetylated radioimmunoassay. Both noradrenaline and VIP elevated intracellular cAMP content concentration dependently, but substance P did not. The response to noradrenaline was blocked by the beta-adrenoceptor antagonist propranolol, but not by the alpha-adrenoceptor antagonist prazosin. Application of the VIP analogue [D-p-Cl-Phe6, Leu17]-VIP decreased the VIP-induced cAMP response. These results demonstrate that striated intralobular duct cells possess beta-adrenoceptors and peptidergic receptors that are coupled to adenylate cyclase and activated by noradrenaline and VIP, respectively. By elevating ductal cAMP content, these agonists may regulate both the electrolyte content of the primary saliva and the secretion of protein(s) from the ducts.

Comparison of brief group therapies for depressed cancer patients receiving radiation treatment.

Although many studies have documented patterns of emotional distress in persons undergoing radiation treatment for cancer, there have been few controlled evaluations of counseling or psychotherapy outcomes with these persons. In this research, the effects of cognitive-behavioral and socially supportive group therapy were evaluated. A total of 72 depressed cancer patients were randomly assigned to one of three conditions--cognitive-behavioral treatment, social support, or a no-treatment control condition. Before and after intervention and at 6-month followup, study participants were individually assessed by using measures of symptom distress. Relative to the comparison group, both the cognitive-behavioral and social support therapies resulted in less depression, hostility, and somatization. The social support intervention also resulted in fewer psychiatric symptoms and reduced maladaptive interpersonal sensitivity and anxiety. It was concluded that both group therapies can reduce symptoms of distress for depressed persons undergoing radiation treatment for cancer. Both forms of therapy resulted in improvements in psychosocial function (compared with no treatment at all), but social support groups demonstrated more changes that were evident at 6-month followup. Further research is needed to evaluate the differential effectiveness of mental health services provided to cancer patients undergoing radiation.

Regulation of fluid secretion and intracellular messengers in isolated rat pancreatic ducts by acetylcholine.

1. We have studied the effects of acetylcholine (ACh) on fluid secretion and intracellular messengers in interlobular ducts isolated from the rat pancreas and maintained in short-term tissue culture. 2. Ductal fluid secretion was measured using micropuncture techniques. Intracellular free calcium ([Ca2+]i) and cyclic AMP concentrations were measured in single ducts using fura-2 microspectrofluorimetry and radioimmunoassay techniques respectively. Changes in the levels of these intracellular messengers were correlated with fluid secretion. 3. ACh stimulated ductal fluid secretion. The dose required for a half-maximal response was about 0.4 microM and maximal secretion was achieved with 10 microM ACh. These effects of ACh were blocked by atropine and by removal of extracellular Ca2+. 4. ACh was about four orders of magnitude less potent as an activator of ductal fluid transport than the hormone secretin; however, the maximal rates of fluid secretion evoked by these two agonists were similar. 5. ACh caused a dose-dependent rise in duct cell [Ca2+]i, but had no effect on cyclic AMP. In contrast, secretin increased duct cell cyclic AMP, but had no effect on [Ca2+]i. 6. The [Ca2+]i response evoked by ACh resulted from both mobilization of intracellular Ca2+ stores and influx of Ca2+ from the extracellular space. 7. The Ca2+ ionophore, ionomycin, mimicked the effect of ACh on ductal [Ca2+]i and fluid secretion. 8. We conclude that ACh stimulates fluid secretion from rat pancreatic duct cells by activating a 'Ca2+ pathway' which is distinct from the well documented 'cyclic AMP pathway' utilized by secretin.

Graphic analysis of superoxide secretion by phagocytic leukocytes at different cell concentrations.

Superoxide secretion by monocytes, macrophages and neutrophils becomes less efficient in a non-linear manner as the cell concentration is increased. This relation holds for cells in suspension or attached to a substrate, elicited into the peritoneum with various agents or from the peripheral circulation, acutely stimulated with particulate or soluble agents, and in experimental animals and in man. The significant observation of this study is that in all these systems, of data from our own studies as well as from the literature, plots of the logarithm of superoxide secreted per cell per unit of time versus the logarithm of the cell concentration were linear with correlation coefficients better than 0.98. The common practice of comparing data on superoxide secretion from experiments performed at different cell concentrations is clearly unsatisfactory. It is suggested that superoxide secretion be expressed in terms of both the slope of the log log plot and the secretion rate at a given cell concentration.

Structural and functional characterization of striated ducts isolated from the rabbit mandibular salivary gland.

Ductal elements within salivary glands are responsible for modifying the electrolyte composition of primary saliva secreted by the acini. To study the mechanism and regulation of the transport processes involved requires a suitable preparation of functional ducts. To this end we have isolated intralobular ducts from rabbit mandibular salivary glands using the technique of tissue dissociation and microdissection. Light and electron microscopy demonstrated that the ducts corresponded ultrastructurally to striated intralobular ducts of the intact gland. Ducts could be maintained in tissue culture on polycarbonate filter rafts for up to 36 h, during which time the ends of the ducts did not usually seal. The overall resting content of ductal adenosine 3',5'-cyclic monophosphate (cyclic AMP) was 16.0 +/- 3.0 fmol mm-1 and increased dose dependently in response to stimulation with the beta-adrenoceptor agonist isoprenaline (10(-9)-10(-4) M; concentration required to produce a half-maximal response, K0.5 = 2.1 x 10(-6) M). The response to isoprenaline was blocked by the antagonist propranolol. Intracellular cyclic AMP content was also raised by the adenylate cyclase activator forskolin and by prostaglandin E2. Acetylcholine (3 x 10(-8)-10(-5) M) caused a dose-dependent and maintained rise in [Ca2+]i (K0.5 = 2.5 x 10(-7) M). This increase in [Ca2+]i could be reversed by the muscarinic antagonist atropine and appeared to result from a combination of mobilization of intracellular Ca2+ stores and entry of Ca2+ from the extracellular fluid. Noradrenaline induced only a very small, mainly transient rise in [Ca2+]i while phenylephrine failed to increase [Ca2+]i at all. Vasoactive intestinal peptide (5 x 10(-7) M) also produced a marginal, maintained rise in [Ca2+]i. Substance P, bombesin, isoprenaline, and prostaglandin E2 did not elevate [Ca2+]i. Application of the calcium ionophore ionomycin induced a substantial maintained rise in [Ca2+]i. Taken together, these results indicate that isolated and cultured striated ducts (i) possess intact beta-adrenoceptors coupled to adenylate cyclase, putative receptors for prostaglandin E2 and muscarinic receptors, and (ii) represent a viable preparation for the study of the transport mechanisms involved in the ductal modification of salivary fluid composition.