Inhibition of pancreatic acinar mitochondrial thiamin pyrophosphate uptake by the cigarette smoke component 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Thiamin is essential for normal metabolism in pancreatic acinar cells (PAC) and is obtained from their microenvironment through specific plasma-membrane transporters, converted to thiamin pyrophosphate (TPP) in the cytoplasm, followed by uptake of TPP by mitochondria through the mitochondrial TPP (MTPP) transporter (MTPPT; product of SLC25A19 gene). TPP is essential for normal mitochondrial function. We examined the effect of long-term/chronic exposure of PAC in vitro (pancreatic acinar 266-6 cells) and in vivo (wild-type or transgenic mice carrying the SLC25A19 promoter) of the cigarette smoke toxin, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), on the MTPP uptake process. Our in vitro and in vivo findings demonstrate that NNK negatively affects MTPP uptake and reduced expression of MTPPT protein, MTPPT mRNA, and heterogenous nuclear RNA, as well as SLC25A19 promoter activity. The effect of NNK on Slc25a19 transcription was neither mediated by changes in expression of transcriptional factor NFY-1 (known to drive SLC25A19 transcription), nor due to changes in methylation profile of the Slc25a19 promoter. Rather, it appears to be due to changes in histone modifications that involve significant decreases in histone H3K4-trimethylation and H3K9-acetylation (activation markers). The effect of NNK on MTPPT function is mediated through the nonneuronal α7-nicotinic acetylcholine receptor (α7-nAChR), as indicated by both in vitro (using the nAChR antagonist mecamylamine) and in vivo (using an α7-nAchR(-/-) mouse model) studies. These findings demonstrate that chronic exposure of PAC to NNK negatively impacts PAC MTPP uptake. This effect appears to be exerted at the level of Slc25a19 transcription, involve epigenetic mechanism(s), and is mediated through the α7-nAchR.

Cerulein hyperstimulation decreases AMP-activated protein kinase levels at the site of maximal zymogen activation.

The premature activation of digestive enzyme zymogens in the pancreatic acinar cell is an important initiating event in acute pancreatitis. We have previously demonstrated that vacuolar ATPase (vATPase) activity is required for zymogen activation. Adenosine monophosphate-activated protein kinase (AMPK) regulates vATPase function in kidney and epididymal clear cells. To determine whether AMPK could affect pancreatitis responses, its effects were first examined in a cellular model of pancreatitis, cerulein-hyperstimulated (100 nM) pancreatic acini. This treatment caused a prominent increase in trypsin and chymotrypsin activities. Pretreatment with AICAR or metformin (AMPK activators) or compound C (an AMPK inhibitor) reduced or increased cerulein-induced zymogen activation, respectively. The association of the vATPase E subunit with membranes, a marker of its activation, tended to be inversely related to AMPK activity (assessed by AICAR and compound C treatments). Cerulein treatment did not change AMPK (α and ß) levels but did lead to an increase in its activation (phosphorylation of Thr172) and induced the time-dependent translocation of the enzyme to a Triton-insoluble compartment. Basal in vivo studies showed that AMPK was widely distributed between membrane and soluble fractions generated by differential centrifugation. After cerulein hyperstimulation, AMPK levels selectively decreased in fractions containing the highest levels of active zymogens. These studies suggest that AMPK activity has a protective role in the pancreatic acinar cell that inhibits zymogen activation in the basal state, and this AMPK effect is reduced during pancreatitis. Therapies that prevent the selective reduction of AMPK in compartments that support zymogen activation could reduce injury during pancreatitis.

A novel protein kinase D inhibitor attenuates early events of experimental pancreatitis in isolated rat acini.

Novel protein kinase C isoforms (PKC δ and ε) mediate early events in acute pancreatitis. Protein kinase D (PKD/PKD1) is a convergent point of PKC δ and ε in the signaling pathways triggered through CCK or cholinergic receptors and has been shown to activate the transcription factor NF-κB in acute pancreatitis. For the present study we hypothesized that a newly developed PKD/PKD1 inhibitor, CRT0066101, would prevent the initial events leading to pancreatitis. We pretreated isolated rat pancreatic acinar cells with CRT0066101 and a commercially available inhibitor Gö6976 (10 µM). This was followed by stimulation for 60 min with high concentrations of cholecystokinin (CCK, 0.1 µM), carbachol (CCh, 1 mM), or bombesin (10 µM) to induce initial events of pancreatitis. PKD/PKD1 phosphorylation and activity were measured as well as zymogen activation, amylase secretion, cell injury and NF-κB activation. CRT0066101 dose dependently inhibited secretagogue-induced PKD/PKD1 activation and autophosphorylation at Ser-916 with an IC(50) ∼3.75-5 µM but had no effect on PKC-dependent phosphorylation of the PKD/PKD1 activation loop (Ser-744/748). Furthermore, CRT0066101 reduced secretagogue-induced zymogen activation and amylase secretion. Gö6976 reduced zymogen activation but not amylase secretion. Neither inhibitor affected basal zymogen activation or secretion. CRT0066101 did not affect secretagogue-induced cell injury or changes in cell morphology, but it reduced NF-κB activation by 75% of maximal for CCK- and CCh-stimulated acinar cells. In conclusion, CRT0066101 is a potent and specific PKD family inhibitor. Furthermore, PKD/PKD1 is a potential mediator of zymogen activation, amylase secretion, and NF-κB activation induced by a range of secretagogues in pancreatic acinar cells.

The emerging role of smoking in the development of pancreatitis.

BACKGROUND/AIMS: Cigarette smoking has been linked to many diseases, including pancreatic cancer and more recently, pancreatitis. METHODS: Electronic searches of primarily PubMed from 1990 to August 2011 were conducted and only articles published in English were reviewed. Original articles and reviews were selected based on screening of article abstracts and their relevance to tobacco smoking, its components, nicotine and its metabolites, and their effects particularly on the pancreas. RESULTS: Smoking may affect the risk of developing chronic pancreatitis or its progression. Smoking may also affect the risk for developing acute pancreatitis. Its effects in pancreatitis appear to be dose dependent and its effects may be alcohol independent but synergize with alcohol. CONCLUSION: Specific constituents of cigarette smoke, including nicotine and its metabolites, could mediate effects on the pancreas.

Reducing extracellular pH sensitizes the acinar cell to secretagogue-induced pancreatitis responses in rats.

BACKGROUND & AIMS: Protease activation within the pancreatic acinar cell is a key early event in acute pancreatitis and may require low pH intracellular compartments. Clinical studies suggest that acidosis may affect the risk for developing pancreatitis. We hypothesized that exposure to an acid load might sensitize the acinar cell to secretagogue-induced pancreatitis. METHODS: Secretagogues (cerulein, carbachol, and bombesin) can induce protease activation in acinar cells at high (100 nmol/L, 1 mmol/L, and 10 micromol/L, respectively) but not at physiologically relevant concentrations. The effects of decreasing extracellular pH (pHe) in early secretagogue-induced pancreatitis (zymogen activation and injury) were examined in rats (1) in vitro with isolated acini and (2) in vivo with an acid challenge. RESULTS: In acini, lowering pHe from 7.6 to 6.8 enhanced secretagogue-induced zymogen activation and injury, but did not affect secretion. For cerulein, this sensitization was seen over a range of concentrations (0.01-100.00 nmol/L). However, reduced pHe alone had no effect on zymogen activation, amylase secretion, or cell injury. We have reported that zymogen activation is mediated by the vacuolar ATPase (vATPase), a proton transporter. vATPase inhibition, using concanamycin (100 nmol/L), blocked the low pHe effects on zymogen activation. An acute acid load given in vivo enhanced cerulein-induced (50 microg/kg) trypsinogen activation and pancreatic edema. CONCLUSION: These studies suggest that acid challenge sensitizes the pancreatic acinar cell to secretagogue-induced zymogen activation and injury and may increase the risk for the development and severity of acute pancreatitis.

Molecular and cellular mechanisms of pancreatic injury.

PURPOSE OF REVIEW: This review focuses on studies from the past year that highlight molecular and cellular mechanisms of pancreatic injury arising from acute and chronic pancreatitis. RECENT FINDINGS: Factors that induce or ameliorate injury as well as cellular pathways involved have been examined. Causative or sensitizing factors include refluxed bile acids, hypercalcemia, ethanol, hypertriglyceridemia, and acidosis. In addition, the diabetes drug exendin-4 has been associated with pancreatitis, whereas other drugs may reduce pancreatic injury. The intracellular events that influence disease severity are better understood. Cathepsin-L promotes injury through an antiapoptotic effect, rather than by trypsinogen activation. In addition, specific trypsinogen mutations lead to trypsinogen misfolding, endoplasmic reticulum stress, and injury. Endogenous trypsin inhibitors and upregulation of proteins including Bcl-2, fibroblast growth factor 21, and activated protein C can reduce injury. Immune cells, however, have been shown to increase injury via an antiapoptotic effect. SUMMARY: The current findings are critical to understanding how causative factors initiate downstream cellular events resulting in pancreatic injury. Such knowledge will aid in the development of targeted treatments for pancreatitis. This review will first discuss factors influencing pancreatic injury, and then conclude with studies detailing the cellular mechanisms involved.

Chronic Nicotine Exposure In Vivo and In Vitro Inhibits Vitamin B1 (Thiamin) Uptake by Pancreatic Acinar Cells.

Thiamin (vitamin B1), a member of the water-soluble family of vitamins, is essential for normal cellular functions; its deficiency results in oxidative stress and mitochondrial dysfunction. Pancreatic acinar cells (PAC) obtain thiamin from the circulation using a specific carrier-mediated process mediated by both thiamin transporters -1 and -2 (THTR-1 and THTR-2; encoded by the SLC19A2 and SLC19A3 genes, respectively). The aim of the current study was to examine the effect of chronic exposure of mouse PAC in vivo and human PAC in vitro to nicotine (a major component of cigarette smoke that has been implicated in pancreatic diseases) on thiamin uptake and to delineate the mechanism involved. The results showed that chronic exposure of mice to nicotine significantly inhibits thiamin uptake in murine PAC, and that this inhibition is associated with a marked decrease in expression of THTR-1 and THTR-2 at the protein, mRNA and hnRNAs level. Furthermore, expression of the important thiamin-metabolizing enzyme, thiamin pyrophosphokinase (TPKase), was significantly reduced in PAC of mice exposed to nicotine. Similarly, chronic exposure of cultured human PAC to nicotine (0.5 µM, 48 h) significantly inhibited thiamin uptake, which was also associated with a decrease in expression of THTR-1 and THTR-2 proteins and mRNAs. This study demonstrates that chronic exposure of PAC to nicotine impairs the physiology and the molecular biology of the thiamin uptake process. Furthermore, the study suggests that the effect is, in part, mediated through transcriptional mechanism(s) affecting the SLC19A2 and SLC19A3 genes.

Tissue Pharmacology of Da-Cheng-Qi Decoction in Experimental Acute Pancreatitis in Rats.

Objectives. The Chinese herbal medicine Da-Cheng-Qi Decoction (DCQD) can ameliorate the severity of acute pancreatitis (AP). However, the potential pharmacological mechanism remains unclear. This study explored the potential effective components and the pharmacokinetic characteristics of DCQD in target tissue in experimental acute pancreatitis in rats. Methods. Acute pancreatitis-like symptoms were first induced in rats and then they were given different doses of DCQD (6 g/kg, 12 g/kg, and 24 g/kg body weight) orally. Tissue drug concentration, tissue pathological score, and inflammatory mediators in pancreas, intestine, and lung tissues of rats were examined after 24 hours, respectively. Results. Major components of DCQD could be found in target tissues and their concentrations increased in conjunction with the intake dose of DCQD. The high-dose compounds showed maximal effect on altering levels of anti-inflammatory (interleukin-4 and interleukin-10) and proinflammatory markers (tumor necrosis factor α and interleukin-6) and ameliorating the pathological damage in target tissues (P < 0.05). Conclusions. DCQD could alleviate pancreatic, intestinal, and lung injury by altering levels of inflammatory cytokines in AP rats with tissue distribution of its components.

Risk factors for pancreatic cancer: underlying mechanisms and potential targets.

PURPOSE OF THE REVIEW: Pancreatic cancer is extremely aggressive, forming highly chemo-resistant tumors, and has one of the worst prognoses. The evolution of this cancer is multi-factorial. Repeated acute pancreatic injury and inflammation are important contributing factors in the development of pancreatic cancer. This article attempts to understand the common pathways linking pancreatitis to pancreatic cancer. RECENT FINDINGS: Intracellular activation of both pancreatic enzymes and the transcription factor NF-κB are important mechanisms that induce acute pancreatitis (AP). Recurrent pancreatic injury due to genetic susceptibility, environmental factors such as smoking, alcohol intake, and conditions such as obesity lead to increases in oxidative stress, impaired autophagy and constitutive activation of inflammatory pathways. These processes can stimulate pancreatic stellate cells, thereby increasing fibrosis and encouraging chronic disease development. Activation of oncogenic Kras mutations through inflammation, coupled with altered levels of tumor suppressor proteins (p53 and p16) can ultimately lead to development of pancreatic cancer. SUMMARY: Although our understanding of pancreatitis and pancreatic cancer has tremendously increased over many years, much remains to be elucidated in terms of common pathways linking these conditions.

Activation of soluble adenylyl cyclase protects against secretagogue stimulated zymogen activation in rat pancreaic acinar cells.

An early feature of acute pancreatitis is activation of zymogens, such as trypsinogen, within the pancreatic acinar cell. Supraphysiologic concentrations of the hormone cholecystokinin (CCK; 100 nM), or its orthologue cerulein (CER), induce zymogen activation and elevate levels of cAMP in pancreatic acinar cells. The two classes of adenylyl cyclase, trans-membrane (tmAC) and soluble (sAC), are activated by distinct mechanisms, localize to specific subcellular domains, and can produce locally high concentrations of cAMP. We hypothesized that sAC activity might selectively modulate acinar cell zymogen activation. sAC was identified in acinar cells by PCR and immunoblot. It localized to the apical region of the cell under resting conditions and redistributed intracellularly after treatment with supraphysiologic concentrations of cerulein. In cerulein-treated cells, pre-incubation with a trans-membrane adenylyl cyclase inhibitor did not affect zymogen activation or amylase secretion. However, treatment with a sAC inhibitor (KH7), or inhibition of a downstream target of cAMP, protein kinase A (PKA), significantly enhanced secretagogue-stimulated zymogen activation and amylase secretion. Activation of sAC with bicarbonate significantly inhibited secretagogue-stimulated zymogen activation; this response was decreased by inhibition of sAC or PKA. Bicarbonate also enhanced secretagogue-stimulated cAMP accumulation; this effect was inhibited by KH7. Bicarbonate treatment reduced secretagogue-stimulated acinar cell vacuolization, an early marker of pancreatitis. These data suggest that activation of sAC in the pancreatic acinar cell has a protective effect and reduces the pathologic activation of proteases during pancreatitis.

Protein kinase C delta-mediated processes in cholecystokinin-8-stimulated pancreatic acini.

OBJECTIVES: To define the role of protein kinase C delta (PKC delta) in acinar cell responses to the hormone cholecystokinin-8 (CCK) using isoform-specific inhibitors and a previously unreported genetic deletion model. METHODS: Pancreatic acinar cells were isolated from (1) rat, and pretreated with a PKC delta-specific inhibitor or (2) PKC delta-deficient and wild type mice. Isolated cells were stimulated with CCK (0.001-100 nmol/L) and cell responses were measured. RESULTS: The PKC delta inhibitor did not affect stimulated amylase secretion from rat pancreatic acinar cells. Cholecystokinin-8 stimulation induced a typical biphasic dose-response curve for amylase secretion in acinar cells isolated from both PKC delta(-/-) and wild type mice, with maximal stimulation at 10-pmol/L CCK. Cholecystokinin-8 (100 nmol/L) induced zymogen and nuclear factor kappaB activation in both PKC delta(-/-) and wild type mice, although it was up to 50% less in PKC delta(-/-). CONCLUSIONS: In contrast to previous studies, this study has used specific and complementary approaches to examine PKC delta-mediated acinar cell responses. We could not confirm that it mediates amylase release but corroborated its role in the early stages of acute pancreatitis.

The novel protein kinase C isoforms -delta and -epsilon modulate caerulein-induced zymogen activation in pancreatic acinar cells.

Isoforms of protein kinase C (PKC) have been shown to modulate some cellular responses such as pathological secretion and generation of inflammatory mediators during acute pancreatitis (AP). We propose that PKC also participates in premature zymogen activation within the pancreatic acinar cell, a key event in the initiation of AP. This hypothesis was examined in in vivo and cellular models of caerulein-induced AP using PKC activators and inhibitors. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA, 200 nM), a known activator of PKC, enhanced zymogen activation at both 0.1 nM and 100 nM caerulein, concentrations which mimic physiological and supraphysiological effects of the hormone cholecystokinin, respectively, in preparations of pancreatic acinar cells. Isoform-specific PKC inhibitors for PKC-delta and PKC-epsilon reduced supraphysiological caerulein-induced zymogen activation. Using a cell-free reconstitution system, we showed that inhibition of PKC-delta and -epsilon, reduced zymogen activation in both zymogen granule-enriched and microsomal fractions. In dispersed acinar cells, 100 nM caerulein stimulation caused PKC-delta and -epsilon isoform translocation to microsomal membranes using cell fractionation and immunoblot analysis. PKC translocation was confirmed with in vivo studies and immunofluorescence microscopy in pancreatic tissues from rats treated with or without 100 nM caerulein. PKC-epsilon redistributed from an apical to a supranuclear region following caerulein administration. The signal for PKC-epsilon overlapped with granule membrane protein, GRAMP-92, an endosomal/lysosomal marker, in a supranuclear region where zymogen activation takes place. These results indicate that PKC-delta and -epsilon isoforms translocate to specific acinar cell compartments and modulate zymogen activation.

Secretion and fluid transport mechanisms in the mammary gland: comparisons with the exocrine pancreas and the salivary gland.

Milk is a complex fluid composed of proteins, sugars, lipids and minerals, in addition to a wide variety of bioactive molecules including vitamins, trace elements and growth factors. The composition of these components reflects the integrated activities of distinct synthetic, secretion and transport processes found in mammary epithelial cells, and mirrors the differing nutritional and developmental requirements of mammalian neonates. Five general pathways have been described for secretion of milk components. With the exception of lipids, which are secreted a unique pathway, milk components are thought to be secreted by adaptations of pathways found in other secretory organs. However little is known about the molecular and cellular mechanisms that constitute these pathways or the physiological mechanisms by which they are regulated. Comparisons of current secretion and transport models in the mammary gland, exocrine pancreas and salivary gland indicate that significant differences exist between the mammary gland and other exocrine organs in how proteins and lipids are packaged and secreted, and how fluid is transported.

Zymogen activation in a reconstituted pancreatic acinar cell system.

Pathological activation of digestive zymogens within the pancreatic acinar cell initiates acute pancreatitis. Cytosolic events regulate this activation within intracellular compartments of unclear identity. In an in vivo model of acute pancreatitis, zymogen activation was detected in both zymogen granule-enriched and microsomal cellular fractions. To examine the mechanism of this activation in vitro, a reconstituted system was developed using pancreatic cytosol, a zymogen granule-enriched fraction, and a microsomal fraction. Addition of cytosol to either particulate fraction resulted in a prominent increase in both trypsin and chymotrypsin activities. The percentage of the pool of trypsinogen and chymotrypsinogen activated was about twofold and sixfold greater, respectively, in the microsomal than in the zymogen granule-enriched fraction. Activation of chymotrypsinogen but not trypsinogen was significantly enhanced by ATP (5 mM) but not by the inactive ATP analog AMP-PNP. The processing of procarboxypeptidase B to its mature form also demonstrated a requirement for ATP and cytosol. E64d, an inhibitor of cathepsin B, a thiol protease that can activate trypsin, completely inhibited trypsin activity but did not affect chymotrypsin activity or carboxypeptidase B generation. These studies demonstrate that both zymogen granule-enriched and microsomal fractions from the pancreas can support cytosol-dependent zymogen activation. A component of the activation of some zymogens, such as chymotrypsinogen and procarboxypeptidase, may depend on ATP but not on trypsin or cathepsin B.

Vacuolar adenosine triphosphatase and pancreatic acinar cell function.

The pathologic activation of proteases within the pancreatic acinar cell is a key initiating event in acute pancreatitis. Past studies have suggested that the generation of a low-pH environment is critical to this process. Vacuolar adenosine triphosphatase (vATPase) is a multiprotein complex that transports protons across cellular membranes. Activation of the vATPase requires assembly of the soluble (V(1)) subunits on the membrane subunits (V(0)). It is found that conditions that cause protease activation in the acinar cell also cause assembly of V(1) on V(0). Further, inhibitors of vATPase block this protease activation. Ethanol and butanol sensitize the acinar cell to cholecystokinin-induced zymogen activation; vATPase inhibitors also blocked this activation. Activation of the vATPase may be central to the pathologic activation of proteases in the acinar cell and may also modulate the sensitizing effects of alcohols.

Vacuolar ATPase regulates zymogen activation in pancreatic acini.

Supramaximal concentrations of cholecystokinin or its analogue caerulein have been shown to stimulate the proteolytic activation of zymogens within the pancreatic acinar cell and initiate acute pancreatitis. Previous studies suggest that a low pH compartment might be required for activation. To test this hypothesis, the effects of agents that modulate intracellular pH on caerulein-induced trypsin and chymotrypsin activation were studied. Pretreatment of pancreatic acini with the proto-ionophore monensin (10 microM) and the weak base chloroquine (40 microM) inhibited activation. Pre-incubation with the vacuolar ATPase (V-ATPase) inhibitors bafilomycin A(1) and concanamycin A also decreased activation in a concentration-dependent manner with 50% inhibition at approximately 50 and 25 nM, respectively. Caerulein stimulation caused a time- and concentration-dependent translocation of soluble V-ATPase V(1) subunits to a membrane fraction, a marker of V-ATPase activation. Carbachol also stimulated translocation at supramaximal concentrations. Elevation of cytosolic Ca(2+) by thapsigargin was sufficient to induce translocation. Thus, stimulation of V-ATPase activity appears to be required for agonist-induced zymogen activation in the pancreatic acinar cell.

A functional interaction between chromogranin B and the inositol 1,4,5-trisphosphate receptor/Ca2+ channel.

Chromogranins A and B (CGA and CGB) are high capacity, low affinity calcium (Ca2+) storage proteins found in many cell types most often associated with secretory granules of secretory cells but also with the endoplasmic reticulum (ER) lumen of these cells. Both CGA and CGB associate with inositol 1,4,5-trisphosphate receptor (InsP3R) in a pH-dependent manner. At an intraluminal pH of 5.5, as found in secretory vesicles, both CGA and CGB bind to the InsP3R. When the intraluminal pH is 7.5, as found in the ER, CGA totally dissociates from InsP3R, whereas CGB only partially dissociates. To investigate the functional consequences of the interaction between the InsP3R and CGB monomers or CGA/CGB heteromers, purified mouse InsP3R type I were fused to planar lipid bilayers and activated by 2 microM InsP3. In the presence of luminal CGB monomers or CGA/CGB heteromers the InsP3R/Ca2+ channel open probability and mean open time increased significantly. The channel activity remained elevated when the pH was changed to 7.5, a reflection of CGB binding to the InsP3R even at pH 7.5. These results suggest that CGB may play an important modulatory role in the control of Ca2+ release from the ER. Furthermore, the difference in the ability of CGA and CGB to regulate the InsP3R/Ca2+ channel and the variability of CGA/CGB ratios could influence the pattern of InsP3-mediated Ca2+ release.

Activation of the inositol 1,4,5-trisphosphate receptor by the calcium storage protein chromogranin A.

Secretory granules of neuroendocrine cells are inositol 1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) stores in which the Ca(2+) storage protein, chromogranin A (CGA), couples with InsP(3)-gated Ca(2+) channels (InsP(3)R) located in the granule membrane. The functional aspect of this coupling has been investigated via release studies and planar lipid bilayer experiments in the presence and absence of CGA. CGA drastically increased the release activity of the InsP(3)R by increasing the channel open probability by 9-fold and the mean open time by 12-fold. Our results show that CGA-coupled InsP(3)Rs are more sensitive to activation than uncoupled receptors. This modulation of InsP(3)R channel activity by CGA appears to be an essential component in the control of intracellular Ca(2+) concentration by secretory granules and may regulate the rate of vesicle fusion and exocytosis.

Three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor at 24 A resolution.

We report here the first three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R). From cryo-electron microscopic images of purified receptors embedded in vitreous ice, a three-dimensional structure was determined by use of standard single particle reconstruction techniques. The structure is strikingly different from that of the ryanodine receptor at similar resolution despite molecular similarities between these two calcium release channels. The 24 A resolution structure of the IP(3)R takes the shape of an uneven dumbbell, and is approximately 170 A tall. Its larger end is bulky, with four arms protruding laterally by approximately 50 A and, in comparison with the receptor topology, probably corresponds to the cytoplasmic domain of the receptor. The lateral dimension at the height of the protruding arms is approximately 155 A. The smaller end, whose lateral dimension is approximately 100 A, has structural features indicative of the membrane-spanning domain. A central opening in this domain, which is occluded on the cytoplasmic half, outlines a pathway for calcium flow in the open state of the channel.