Extracellular vesicles (EVs) are associated with intercellular communications, immune responses, viral pathogenicity, cardiovascular diseases, neurological disorders, and cancer progression. EVs deliver proteins, metabolites, and nucleic acids into recipient cells to effectively alter their physiological and biological response. During their transportation from the donor to the recipient cell EVs face differential ionic concentrations, which can be detrimental to their integrity and impact their cargo content. EVs are known to possess ion channels and transporters in their membrane but neither the function nor the role of these channels in EVs is known. In this study, we discover a functional calcium-activated large-conductance potassium channel (BKCa) in the membrane of EVs. Furthermore, we establish that BKCa is essential for the structural and functional integrity of EVs. Together, these findings establish the critical role of ion channels such as BKCa in functioning as gatekeepers and maintaining EV-mediated signaling.

The choroid plexus (CP) epithelium plays a major role in the production of cerebrospinal fluid (CSF). A polarized cell line, the porcine CP-Riems (PCP-R) line, which exhibits many of the characteristics of the native epithelium, was used to study the effect of activation of the transient receptor potential vanilloid 4 (TRPV4) cation channel found in the PCP-R cells as well as in the native epithelium. Ussing-style electrophysiological experiments showed that activation of TRPV4 with a specific agonist, GSK1016790A, resulted in an immediate increase in both transepithelial ion flux and conductance. These changes were inhibited by either of two distinct antagonists, HC067047 or RN1734. The change in conductance was reversible and did not involve disruption of epithelial junctional complexes. Activation of TRPV4 results in Ca 2+ influx, therefore, we examined whether the electrophysiological changes were the result of secondary activation of Ca 2+ -sensitive channels. PCP-R cells contain two Ca 2+ -activated K + channels, the small conductance 2 (SK2) and the intermediate conductance (IK) channels. Based on inhibitor studies, the former is not involved in the TRPV4-mediated electrophysiological changes whereas one of the three isoforms of the IK channel (KCNN4c) may play a role in the apical secretion of K + . Blocking the activity of this IK isoform with TRAM34 inhibited the TRPV4-mediated change in net transepithelial ion flux and the increased conductance. These studies implicate TRPV4 as a hub protein in the control of CSF production through stimulation by multiple effectors resulting in transepithelial ion and subsequent water movement.

The BK potassium channel contributes to K + flow and the electrical behavior of many cell types: including pancreatic β‐cells and cells in the hypothalamic‐pituitary‐adrenal axis. Mice made null for the gene ( Kcnma1, slo‐1 ) producing the BK channel exhibit numerous deficits in physiological functions, but also show resistance toward developing obesity on a high fat diet (Illison J, et al, Diabetes 2016 db160245). Initial breeding pairs lacking a single allele of Kcnma1 (BK het ) in a C57BL/6J background strain were obtained from Dr. Andrea Meredith at the University of Maryland. A colony derived by breeding these heterozygous mice had litter sizes of ~8 pups, 53% male. For the period of maternal care (P0 – P21) pup death peaked at P1 with a second less severe interval of pup death peaking near P13. The later deaths (~P13) of pups null for Kcnma1 (BK KO ) were greater than Mendelian expectations. Pup death at ~P1 was twice as likely during the 20 month construction of a building adjacent to the animal facility compared with the quiescent period after cessation of construction. Births also were not consistent with Mendelian predictions, indicating a specific prenatal disadvantage for female BK KO mice in the quiescent period and for female BK het mice during construction, a change likely induced by this environmental stressor. Death of colony mice after weaning was rare (~0.5%). After weaning, weight gain was lower for BK KO mice compared with wild‐type littermates: male BK KO mice were 5 g smaller and female BK KO mice were 4 g smaller. Lower weight gain for BK KO mice compared to wild‐type occurred in part due to an event of weight loss during the early post‐weaning period, between weeks 3 to 9. Interestingly, wild‐type mice as well as male BK KO mice were ~1 g larger during construction compared with mice during quiescence. Body composition determined by quantitative magnetic resonance (QMR) indicated a higher fat proportion for wild‐type female mice compared with males, as well as a higher hydration ratio (total water mass – free water mass/lean mass). Comparison of lean, fat, and water components for female and male mice suggested an interrelationship that governs changes to body composition. Both male and female BK KO mice showed higher fat proportions than wild‐type, with the female BK KO mice exhibiting a larger variation in fat content and male BK KO mice having a higher hydration ratio than wild‐type. Together these results indicate that BK KO mice suffered disadvantages that led to prenatal and perinatal death. A metabolic difference likely related to glucose handling led to the smaller body size and distinct composition with higher fat for BK KO mice, suggesting a diversion of energy supplies from growth to fat storage. Support or Funding Information [NIH DK65845; WSU‐BSoM Seed Grant]

Big conductance potassium (BK) channels contribute to K+ flow and electrical behavior in many cell types. Mice made null for the gene (Kcnma1) producing the BK channel (BKKO) exhibit numerous deficits in physiological functions. Breeding mice lacking a single allele of Kcnma1 (C57BL/6J background) had litter sizes of approximately eight pups. For the period of maternal care (P0-P21), pup deaths peaked at P1 with a second less severe interval of death peaking near P13. Early deaths were twice as likely during a 20-month period of building construction compared with the quiescent period after cessation of construction. Births during construction were not consistent with Mendelian predictions indicating the likelihood of a specific disadvantage induced by this environmental stressor. Later BKKO pup deaths (similar to P13) also were more numerous than Mendelian expectations. After weaning, weight gain was slower for BKKO mice compared with wild-type littermates: 5 g less for male BKKO mice and 4 g less for female BKKO mice. Body composition determined by quantitative magnetic resonance indicated a higher fat proportion for wild-type female mice compared with males, as well as a higher hydration ratio. Both male and female BKKO mice showed higher fat proportions than wild-type, with female BKKO mice exhibiting greater variation. Together, these results indicate that BKKO mice suffered disadvantages that lead to prenatal and perinatal death. A metabolic difference likely related to glucose handling led to the smaller body size and distinct composition for BKKO mice, suggesting a diversion of energy supplies from growth to fat storage.

We evaluated the conductances for ion flow across the cellular andparacellular pathways offlounder intestine using microelectrode techniques and ion-replacement studies. Apical membrane conductance properties are dominated by the presence of Ba-sensitive K channels . An elevated mucosal solution K concentration, [K]m, depolarized the apical membrane potential (0a) and, at [K]m < 40 mM, the K dependence of 0. was abolished by 1-2 mM mucosal Ba . The basolateral membrane displayed Cl conductance behavior, as evidenced by depolarization of the basolateral membrane potential (4/b) with reduced serosal Cl concentrations, [Cl],. 9'b was unaffected by changes in [K], or [Na],. From the effect of mucosal Ba on transepithelial K selectivity, we estimated that paracellular conductance (GP) normally accounts for 96% of transepithelial conductance (G,) . The high G, attenuates the contribution of the cellular pathway to 4Gt while permitting the apical K and basolateral Cl conductances to influence the electrical potential differences across both membranes . Thus, 0. and yob (-60 mV, inside negative) lie between the equilibrium potentials for K (76 mV) and Cl (40 mV), thereby establishing driving forces for K secretion across the apical membrane and Cl absorption across the basolateral membran6. Equivalent circuit analysis suggests that apical conductance (G. a 5 mS/cm2) is sufficient to account for the observed rate of K secretion, but that basolateral conductance (Gb 29 1 .5 mS/cm2) would account for only 50% of net Cl absorption . This, together with our failure to detect a basolateral K conductance, suggests that CI absorption across this barrier involves KCI co-transport .

Aldosterone (aldo) and epinephrine (epi) activate electrogenic K+ secretion in distal colon via a cellular mechanism involving apical membrane K+ channels and basolateral Cl- channels. Epi [10 μM] activated a short-circuit current (epiIsc) in ex vivo mouse distal colon (C57BL/6) consistent with transient Cl- secretion and sustained K+ secretion. Prior addition of peptide-YY [0.3 μM] suppressed transient epiIsc revealing sustained epiIsc within 5 min after epi addition. Aldo [1 μM] activated a sustained Isc consistent with K+ secretion after a delay of ~20 min; subsequent epi addition further stimulated Isc in a non-additive manner suggesting that epi alone produced maximal K+ secretion. Secretory activation also was measured in age-matched litter-mates lacking the slo-gene product (BK-knockout). epiIsc in BKKO distal colon was a small transient with a steady-state similar to basal, and aldoIsc remained similar to pre-stimulation Isc. Addition of the BK blockers paxilline or iberiotoxin (IbTx) inhibited epiIsc in wild-type colon supporting a requirement for this channel in the apical membrane. Low sensitivity to IbTx suggests involvement of the β-subunits kcnmb1 or kcnmb4. Basal Isc was more positive in BKKO colon than wildtype consistent with Cl- secretion. Addition of the Ca++-activated Cl- channel blocker CaCCinh-A01 [30 μM] in wild-type increased basalIsc with a resulting Isc similar to basalIsc in BKKO. Partial inhibition of basalIsc by the CFTR Cl- channel blocker CFTRinh-172 [30 μM] suggests an unmasking of basal Cl- secretion in BKKO colon. These results indicate a near complete dependence of electrogenic K+ secretion on the presence of the BK K+ channel in the apical membrane of the mouse distal colonic epithelium. [WSU-BSoM Seed Grant]

Aldosterone (aldo) activates electrogenic K+ secretion in guinea pig distal colon via a mechanism requiring protein synthesis. Channel blocker sensitivity supported conductive apical K+ exit and basolateral Cl‐ exit during aldo stimulation, similar to adrenergic activation. Paxilline [1 μM], BK channel (KCa1.1, Kcnma1) blocker, inhibited ~50% of short‐circuit current (Isc) and transepithelial conductance (Gt) activated by aldo; and, CaCCinh‐A01 [30 μM], Ca++‐activated Cl‐ channel blocker, eliminated aldoIsc and decreased aldoGt. Signaling for K+ secretion by aldo involved cAMP and serum‐glucocorticoid protein kinase (sgk) as indicated by elimination of aldoIsc with inhibition of soluble adenylyl cyclase by KH7 [30 μM] or sgk by GSK‐650394 [10 μM]. Elimination of aldoIsc by inhibiting dynamin (dyngo‐4a [30 μM]) or clathrin (pitstop‐2 [20 μM]) indicated a requirement for clathrin dependent endocytosis during aldo signaling. Manipulating the ubiquitylation cycle by inhibiting ubiquitin‐E1 ligase (UBEI‐41 [50 μM]) also inhibited aldoIsc; and, disrupting membrane localization of small GTPases such as ras, rho, or rab with farnesyltiosalicylate [10 μM] abolished aldoIsc. Dependence on microtubular transport was supported by loss of aldoIsc with inhibiting dynein (ciliobrevin‐D [10 μM]). Together these results support an aldosterone signaling mechanism for activating electrogenic K+ secretion involving cAMP, sgk, endocytosis, and microtubular transport. [NIH DK65845]

A variety of hormones and neurotransmitters activate electrogenic K+ secretion in guinea pig distal colon, generally together with Cl− secretion. Blockers of BK channels (KCa1.1, Kcnma1), iberiotoxin (IbTx) and paxilline, inhibited the short‐circuit current (Isc) associated with K+ secretion. Consistent with this K+ secretion occurring via apical membrane BK, mucosal addition of IbTx inhibited epinephrine (epi) activation with an IC50 for Isc (epiIsc) and for transepithelial conductance (epiGt) of ~200 nM. However, maximal inhibition was only ~50%. Mucosally added paxilline [10 μM] also inhibited epiIsc and epiGt by ~50%. IbTx and paxilline each inhibited Isc activated by mucosal ATP, supporting apical BK as an absolute requirement for this K+ secretion. Sensitivity to IbTx and paxilline demonstrated K+ secretion during activation of Cl− secretion by prostaglandin‐E2 and a cholinergic agonist. Distal colonic epithelial cells expressed BKα mRNA with the ZERO splice variant and 3 splice variants for the C‐terminus. These cells also expressed the regulatory β‐subunits BKβ1 and BKβ4. Immuno‐localization demonstrated BKα in apical and basolateral membranes of surface and crypt cells. Together these results support a cellular mechanism for electrogenic K+ secretion involving activation of apical membrane BK, but epi activated K+ secretion also required opening of other K+ channel types. [NIH DK65845]

Secretagogues acting at a variety of receptor types activate electrogenic K(+) secretion in guinea pig distal colon, often accompanied by Cl(-) secretion. Distinct blockers of K(Ca)1.1 (BK, Kcnma1), iberiotoxin (IbTx), and paxilline inhibited the negative short-circuit current (I(sc)) associated with K(+) secretion. Mucosal addition of IbTx inhibited epinephrine-activated I(sc) ((epi)I(sc)) and transepithelial conductance ((epi)G(t)) consistent with K(+) secretion occurring via apical membrane K(Ca)1.1. The concentration dependence of IbTx inhibition of (epi)I(sc) yielded an IC(50) of 193 nM, with a maximal inhibition of 51%. Similarly, IbTx inhibited (epi)G(t) with an IC(50) of 220 nM and maximal inhibition of 48%. Mucosally added paxilline (10 μM) inhibited (epi)I(sc) and (epi)G(t) by ∼50%. IbTx and paxilline also inhibited I(sc) activated by mucosal ATP, supporting apical K(Ca)1.1 as a requirement for this K(+) secretagogue. Responses to IbTx and paxilline indicated that a component of K(+) secretion occurred during activation of Cl(-) secretion by prostaglandin-E(2) and cholinergic stimulation. Analysis of K(Ca)1.1α mRNA expression in distal colonic epithelial cells indicated the presence of the ZERO splice variant and three splice variants for the COOH terminus. The presence of the regulatory β-subunits K(Ca)β1 and K(Ca)β4 also was demonstrated. Immunolocalization supported the presence of K(Ca)1.1α in apical and basolateral membranes of surface and crypt cells. Together these results support a cellular mechanism for electrogenic K(+) secretion involving apical membrane K(Ca)1.1 during activation by several secretagogue types, but the observed K(+) secretion likely required the activity of additional K(+) channel types in the apical membrane.

Epinephrine (epi) activated Cl and K secretion (sec) in isolated mucosa from guinea pig distal colon, measured as short‐circuit current. These components of the response were distinguished by inhibition of Cl sec with a β2 antagonist (ICI‐118551) that spared K sec; propranolol inhibited both. Interfering with endocytosis prolonged the transient Cl sec and inhibited sustained K sec. Both the dynamin inhibitor dynasore and the clathrin‐coated‐pit blocker mono‐dansylcadaverine produced this response. Dynasore also inhibited the sustained epi‐induced increase in mucosal cAMP, while leaving the transient cAMP increase intact. Manipulating the ubiquitylation cycle by inhibiting ubiquitin‐E1 ligase (UBEI‐41, BioGenova) also prolonged the epi‐induced Cl sec transient and slowed the onset of K sec. Conversely, inhibiting deubiquitylases (PR‐619, LifeSensors) alone stimulated K sec, while blunting Cl sec and producing a non‐additive increase in K sec with epi. Disruption of the actin (latrunculin B, cytochalasin D) and microtubule (nocodazole) cytoskeleton inhibited epi‐induced K sec without altering Cl sec. Together these results support an activation process involving internalization of β2‐adrenergic receptors that terminates signaling for Cl sec, and β‐adrenergic stimulation of K sec that requires receptor internalization prior to activation of the sustained cAMP signaling cascades. [NIH DK65845]

Epinephrine (epi) activates transient Cl secretion (sec) and sustained K sec across isolated distal colonic mucosa of guinea pig. The Ca-activated Cl channel inhibitor CaCCinh-A01 [30μM] reduced electrogenic K sec, detected as short-circuit current (Isc), consistent with basolateral Cl channels providing an exit pathway for Cl entering via Na/K/2Cl-cotransporters. CaCCinh-A01 inhibited both Isc and transepithelial conductance in a concentration dependent manner, IC50 = 6.3μM. GlyH-101, another Cl channel inhibitor, also reduced K secretory Isc (IC50 = 9.4μM). Epi activated whole-cell Cl current in isolated intact colonic crypts. This epi-activated Cl current also was inhibited by CaCCinh-A01 or GlyH-101. In contrast to K sec, CaCCinh-A01 augmented epi-activated Cl secretory Isc as well as PGE2-activated Cl sec. Synergistic Cl sec activated by cholinergic/PGE2 stimulation was insensitive to CaCCinh-A01. Colonic expression of Tmem16A, a Ca-activated Cl channel, was supported by RT-PCR of Tmem16A-mRNA, immuno-blot with Tmem16A-antibodies, and immuno-reactivity in lateral membranes of epithelial cells. Alternative splices of Tmem16A were detected for exons involved in channel activation. Inhibition of K sec and augmentation of Cl sec by CaCCinh-A01 supports a common colonic cell model for these two ion secretory processes with basolateral membrane Cl channels that contribute to production of electrogenic K sec and limiting Cl sec. Maximal Cl sec occurs only for synergistic activation mechanisms that inactivate these basolateral membrane Cl channels. [NIH DK65845]

Adrenaline activates transient Cl- secretion and sustained K+ secretion across isolated distal colonic mucosa of guinea-pigs. The Ca2+-activated Cl- channel inhibitor CaCCinh-A01 (30 mu m) significantly reduced electrogenic K+ secretion, detected as short-circuit current (I-sc). This inhibition supported the cell model for K+ secretion in which basolateral membrane Cl- channels provide an exit pathway for Cl- entering the cell via Na+-K+-2Cl- cotransporters. CaCCinh-A01 inhibited both I-sc and transepithelial conductance in a concentration-dependent manner (IC50 = 6.3 mu m). Another Cl- channel inhibitor, GlyH-101, also reduced sustained adrenaline-activated I-sc (IC50 = 9.4 mu m). Adrenaline activated whole-cell Cl- current in isolated intact colonic crypts, confirmed by ion substitution. This adrenaline-activated whole-cell Cl- current was also inhibited by CaCCinh-A01 or GlyH-101. In contrast to K+ secretion, CaCCinh-A01 augmented the electrogenic Cl- secretion activated by adrenaline as well as that activated by prostaglandin E-2. Synergistic Cl- secretion activated by cholinergic/prostaglandin E-2 stimulation was insensitive to CaCCinh-A01. Colonic expression of the Ca2+-activated Cl- channel protein Tmem16A was supported by RT-PCR detection of Tmem16A mRNA, by immunoblot with a Tmem16A antibody, and by detection of immunofluorescence in lateral membranes of epithelial cells. Alternative splices of Tmem16A were detected for exons that are involved in channel activation. Inhibition of K+ secretion and augmentation of Cl- secretion by CaCCinh-A01 support a common colonic cell model for these two ion secretory processes, such that activation of basolateral membrane Cl- channels contributes to the production of electrogenic K+ secretion and limits the rate of Cl- secretion. Maximal physiological Cl- secretion occurs only for synergistic activation mechanisms that close these basolateral membrane Cl- channels.

Epinephrine (epi) stimulated ion secretion in isolated mucosa from guinea pig distal colon, measured as short‐circuit current. Epi responses included transient Cl (0~5 min) and sustained K secretion (sec). ICI‐118551 (β2‐selective antagonist) inhibited Cl sec (Kd = 2.7 nM), even though inhibition of K sec required combined β1/β2 antagonism. Co‐immunoprecipitation of β1 and β2 adrenergic receptors (βAdrR) provided support for this combined action. Both β1AdrR and β2AdrR also co‐IPed with arrestin‐2, suggesting an alternate signaling path. Whereas dideoxyadenosine (ddAdo; inhibits transmembrane adenylyl cyclase, tmAC) inhibited only Cl sec, KH7 (inhibits soluble adenylyl cyclase, sAC) inhibited only K sec. Consistent with bicarbonate dependence for sAC, sustained K sec was 42% smaller in HEPES buffered bathing media (CO2/HCO3 free), without altering transient Cl sec. Immunoblot for sAC indicated a presence of 54 and 74 kDa bands, consistent with sAC isoforms found in other tissues. ddAdo suppressed the biphasic epi activation of cAMP levels; KH7 left the early transient cAMP unaltered but lowered sustained levels; early cAMP transients remained in the absence of bicarbonate with sustained levels lowered. Thus, β2AdrR activation produced Cl sec via a transient cAMP elevation from tmAC, while β1AdrR/β2AdrR activation produced K sec via a sustained cAMP from sAC that was bicarbonate dependent. [NIH DK65845]

Adrenergic stimulation of isolated guinea pig distal colonic mucosa produced transient Cl(-) and sustained K(+) secretion. Transient short-circuit current (I(sc)) depended on beta(2)-adrenergic receptors (beta(2)-AdrR), and sustained I(sc) relies on a beta(1)-AdrR/beta(2)-AdrR complex. Epinephrine (epi) increased cAMP content with a biphasic time course similar to changes in epi-activated I(sc) ((epi)I(sc)). Inhibition of transmembrane adenylyl cyclases (tmACs) reduced peak (epi)I(sc) and cAMP to near zero without decreasing sustained (epi)I(sc), consistent with cAMP from tmAC signaling for only Cl(-) secretion. Inhibition of soluble adenylyl cyclase (sAC) reduced sustained (epi)I(sc) and cAMP to near zero without decreasing peak (epi)I(sc) or cAMP, consistent with cAMP from sAC signaling for K(+) secretion. Sensitivity to phosphodiesterase (PDE) inhibitors and peptide YY (PYY) stimulation further supported separate signaling for the two components. PDE3 or PDE4 inhibitors enhanced peak (epi)I(sc) but not sustained (epi)I(sc), consistent with these PDEs as part of the beta(2)-AdrR signaling domain. PYY suppressed peak (epi)I(sc) in a pertussis toxin (PTx)-sensitive manner, supporting Galpha(i)-dependent inhibition of tmACs producing cAMP for Cl(-) secretion. Since PYY or PTx did not alter sustained (epi)I(sc), signaling for K(+) secretion occurred via a Galpha(i)-independent mechanism. Presence of multiple sAC variants in colonic epithelial cells was supported by domain-specific antibodies. Responses to specific activators and inhibitors suggested that protein kinase A was not involved in activating peak or sustained components of (epi)I(sc), but the cAMP-dependent guanine nucleotide exchange factor, Epac, may contribute. Thus beta-adrenergic activation of electrogenic Cl(-) and K(+) secretion, respectively, required tmAC- and sAC-dependent signaling pathways.

Adrenergic stimulation of electrogenic K+ secretion in isolated mucosa from guinea pig distal colon required activation of two beta-adrenergic receptor subtypes (beta-AdrR). Addition of epinephrine (epi) or norepinephrine (norepi) to the bathing solution of mucosae in Ussing chambers increased short-circuit current (Isc) and transepithelial conductance (Gt), consistent with this cation secretion. A beta-adrenergic classification was supported by propranolol antagonism of this secretory response and the lack of effect by the alpha-AdrR antagonists BE2254 (alpha1-AdrR) and yohimbine (alpha2-AdrR). Subtype-selective antagonists CGP20712A (beta1-AdrR), ICI-118551 (beta2-AdrR), and SR59320A (beta3-AdrR) were relatively ineffective at inhibiting the epi-stimulated Isc response. In combination, CGP20712A and ICI-118551 inhibited the response, which supported a synergistic action by beta1-AdrR and beta2-AdrR. Expression of mRNA for both beta1-AdrR and beta2-AdrR was indicated by RT-PCR of RNA from colonic epithelial cells. Protein expression was indicated by immunoblot showing bands at molecular weights consistent with monomers and oligomers. Immunoreactivity (ir) for beta1-AdrR and beta2-AdrR was prominent in basolateral membranes of columnar epithelial cells in the crypts of Lieberkühn as well as intercrypt surface epithelium. Cells in the pericryptal sheath also had beta1-AdrR(ir) but did not have discernable beta2-AdrR(ir). The adrenergic sensitivity of K+ secretion measured by Isc and Gt was relatively low as indicated by EC(50)s of 41 +/- 7 nM for epi and 50 +/- 14 nM for norepi. Adrenergic activation of electrogenic K+ secretion required the involvement of both beta1-AdrR and beta2-AdrR, occurring with an agonist sensitivity reduced compared with reported values for either receptor subtype.

Cl and K secretion measured as short‐circuit current (Isc) in isolated mucosa from guinea pig distal colon were stimulated by epinephrine (epi). Cl secretory Isc was transient lasting ~5min, while the negative Isc consistent with K secretion persisted indefinitely. 2'5′‐dideoxyadenosine (ddAdo; inhibits adenylyl cyclase, AC) reduced Cl sec whereas rolipram (inhibits phosphodiesterase) caused an increase; neither altered K sec. Peptide‐YY (PYY; acts via receptor inhibiting AC) reduced Cl sec without altering K sec. Pertussis toxin (inhibits G‐protein‐αi) eliminated PYY inhibition. cAMP levels in colonic mucosa increased at 30s after epi followed by a decrease at 10min, but not to control levels. Forskolin [0.1μM] (stimulates AC and K sec) increased cAMP comparable to epi 10min. PYY inhibited the rise in cAMP at 30s epi; ddAdo blunted the rise in cAMP. 8Br‐cAMP [100μM] stimulated K sec, and at 1mM increased Cl sec. Signaling could act via the cAMP‐dependent effectors protein kinase A (PKA) or Epac. H‐89 (inhibits PKA) reduced Cl sec, but not K sec. 8CPT‐2Me‐cAMP (Epac selective analog) partially stimulated K sec at 100μM. Epi did not activate Rap‐1 (Epac sensitive G‐protein). Thus, β‐adrenergic stimulation activated Cl secretion via a cAMP dependent pathway involving PKA, and activated K secretion via a pathway independent of PKA and Rap1 but possibly involving cAMP‐dependent exchange factor Epac. [NIH DK65845]