Glycosylation defects activate filamentous growth Kss1 MAPK and inhibit osmoregulatory Hog1 MAPK

The yeast filamentous growth (FG) MAP kinase (MAPK) pathway is activated under poor nutritional conditions. We found that the FG‐specific Kss1 MAPK is activated by a combination of an O‐glycosylation defect caused by disruption of the gene encoding the protein O‐mannosyltransferase Pmt4, and an N‐glycosylation defect induced by tunicamycin. The O‐glycosylated membrane proteins Msb2 and Opy2 are both essential for activating the FG MAPK pathway, but only defective glycosylation of Msb2 activates the FG MAPK pathway. Although the osmoregulatory HOG (high osmolarity glycerol) MAPK pathway and the FG MAPK pathway share almost the entire upstream signalling machinery, osmostress activates only the HOG‐specific Hog1 MAPK. Conversely, we now show that glycosylation defects activate only Kss1, while activated Kss1 and the Ptp2 tyrosine phosphatase inhibit Hog1. In the absence of Kss1 or Ptp2, however, glycosylation defects activate Hog1. When Hog1 is activated by glycosylation defects in ptp2 mutant, Kss1 activation is suppressed by Hog1. Thus, the reciprocal inhibitory loop between Kss1 and Hog1 allows only one or the other of these MAPKs to be stably activated under various stress conditions.     

Comparative effects of cytochalasin D on the protein discharge induced by α- and β-adrenergic or cholinergic agonists in rat exorbital lacrimal glands

Cytochalasin D altered the kinetics of peroxidase and radiolabeled protein discharge from rat exorbital lacrimal glands in vitro, in response to various secretagogues. The changes were different with each inducer. The discharge due to isoproterenol was immediately inhibited by 95%; the discharge evoked by noradrenaline via α-adrenergic receptors was progressively reduced and was inhibited by 50% after 30 min, whereas that evoked by carbachol was not influenced during the initial discharge period and was diminished by only 30% after 30 min. When calcium was removed from the incubation medium, the secretory responses were lowered and the inhibitory effect of cytochalasin D was still observed. The rate of protein discharge inhibition was related to the dose and was maximal with 2·10^?6 M cytochalasin D when the discharge resulted from cholinergic, α- or β-adrenergic or dibutyryl cAMP stimulation. Cytochalasin D did not impair cellular energetics nor other stimulations induced through muscarinic or adrenergic receptors. Cytochalasin D effects could be related to interaction with actin, leading to the inhibition of the release of proteins into the incubation medium following the activation of the adrenergic system.     

Chimeric muscarinic cholinergic: beta-adrenergic receptors that activate Gs in response to muscarinic agonists

The M1-muscarinic cholinergic receptor (M1AChR) stimulates the release of inositol phosphates (IPs) but does not activate adenylyl cyclase. The beta-adrenergic receptor (beta-AR) stimulates adenylyl cyclase but has no effect on IP release. Amino acid sequences corresponding to the second (I2) and third (I3) intracellular loops of the turkey erythrocyte beta-AR and a 12-amino acid segment near the N-terminal end of the I3 region were substituted into the corresponding regions of the human M1AChR. Chimeric receptors that contained either the entire I3 loop or the N-terminal dodecapeptide of that loop both mediated the 2-4-fold stimulation of adenylyl cyclase activity in membrane fractions of COS, A293, or Sf9 cells in response to carbachol. These chimeric receptors also retained the ability to stimulate IP release to the same extent as did the M1AChR. In COS cells transfected with the I3 chimeric receptor, the EC50 for carbachol was approximately 7 microM for the stimulation of adenylyl cyclase and approximately 2 microM for the release of IP; M1AChR-mediated IP release displayed an EC50 of approximately 0.2 microM. Substitution of the I2 region of the beta-AR into the M1AChR did not by itself alter selectivity for signaling. However, the I2+I3 and I2+dodecapeptide combined replacements stimulated adenylyl cyclase fully and caused at most 25% of the maximal stimulation of IP release observed with the M1AChR. Thus, a small region in the third cytoplasmic loop can alter the G proteins to which a receptor is coupled, but interaction among loops is evidently involved in fully determining G protein selectivity.     

Effects of cholinergic and α-adrenergic agonists on the monovalent ion content of rat submandibular gland acinar cells studied by X-ray microanalysis

 The effects of cholinergic and α-adrenergic stimulation (in vivo and in vitro) on the monovalent ion content of rat submandibular gland acinar cells were evaluated at the subcellular level by X-ray microanalysis. Fragments of glands or enzymatically dispersed acini were slam-frozen and cut into ultrathin cryosections. Spectra were collected from secretory granules, nucleus, the basal cytoplasm containing endoplasmic reticulum and the apical cytoplasm identified between secretory granules. No significant changes in Na and Cl content were observed after the isolation of acini, but the K concentration decreased compared with cells from in situ glands. The Cl and K content in all four compartments studied decreased significantly after cholinergic stimulation both in vivo and in vitro but in a more restricted fashion after α-adrenergic stimulation. Our findings indicate that: (1) the physiological mechanisms regulating the monovalent ion composition of submandibular cells are relatively well preserved in isolated acinar cells; (2) the results from in vivo experiments are in good agreement with those from in vitro experiments; and (3) the effects of cholinergic and α-adrenergic stimulation on the K⁺ and Cl^–efflux at the subcellular level are similar but the response is generally less with α-adrenergic stimulation. Accepted: 24 April 1997     

Cholinergic agonists transactivate EGFR and stimulate MAPK to induce goblet cell secretion

Conjunctival goblet cells are the primarysource of mucins in the mucous layer, the innermost layer of the tearfilm. Conjunctival goblet cell mucin secretion is under neural controlbecause exogenous addition of parasympathetic agonists stimulatesgoblet cell secretion. To elucidate the intracellular signal pathwaysused by cholinergic agonists to stimulate goblet cell mucin secretion,we determined whether p42/p44 mitogen-activated protein kinase (MAPK)is activated during cholinergic agonist-stimulated mucin secretion. Ratconjunctiva was removed, preincubated with or without antagonists, andstimulated with the cholinergic agonist carbachol (10⁴M). Carbachol statistically significantly stimulated thephosphorylation of MAPK in a time- and concentration-dependent manner.U-0126, an inhibitor of MAPK activation, completely inhibited both the activation of MAPK and goblet cell secretion stimulated by carbachol. The M₁ muscarinic antagonist pirenzepine, theM₂ muscarinic antagonist gallamine, and theM₁/M₃ muscarinic receptor antagonistN-(3-chloropropyl)-4-piperidinyl diphenylacetate (4-DAMP)also inhibited carbachol-stimulated MAPK activation. Increasing theintracellular Ca²⁺ concentration with a Ca²⁺ionophore increased MAPK activation, and chelation of extracellular Ca²⁺ inhibited carbachol-stimulated activation. Carbacholalso increased tyrosine phosphorylation of Pyk2, p60Src, and theepidermal growth factor receptor (EGFR). The Src inhibitor PP1 and theEGFR inhibitor AG-1478 completely inhibited carbachol-stimulated MAPKactivation. AG-1478 also inhibited goblet cell secretion. We concludethat carbachol transactivates the EGFR to activate MAPK, leading to conjunctival goblet cell secretion. In addition, carbachol also activates Pyk2 and p60Src that could play a role in the transactivation of the EGFR.

     

Methylprednisolone and isoproterenol inhibit airway smooth muscle proliferation by separate and additive mechanisms

To determine whether glucocorticoids and β-adrenoceptor agonists act independently to inhibit airway smooth muscle (ASM) proliferation, the present study investigated the effects of methylprednisolone (MP) and isoproterenol (ISO) alone and in combination on leukotriene D₄-induced ASM proliferation. MP and ISO had no effect on unstimulated ASM cell growth. In contrast, MP and ISO demonstrated dose-dependent inhibition of LTD₄-induced proliferation, and the inhibitory effect was additive for combinations of MP and ISO. The competitive cAMP receptor antagonist, Rp-cAMPS, ablated the ISO-induced inhibition but had no affect on the inhibitory response to MP. In cells exposed to both ISO and MP, Rp-cAMPS attenuated the growth inhibition to levels achieved by MP alone. Accordingly, these findings demonstrate that glucocorticoids and β-adrenergic agonists inhibit LTD₄-induced ASM proliferation, and that their inhibitory effects are mediated by different signaling pathways.     

Cholecystokinin and EGF activate a MAPK cascade by different mechanisms in rat pancreatic acinar cells

The effects of activating the G_qprotein-coupled cholecystokinin (CCK) receptor on differentproteins/signaling molecules in the mitogen-activated protein kinase(MAPK) cascade in pancreatic acinar cells were analyzed and comparedwith the effects of activating the tyrosine kinase-coupled epidermalgrowth factor (EGF) receptor. Both EGF and CCK octapeptide rapidlyincreased the activity of the MAPKs [extracellular signal-regulatedkinase (ERK) 1 and ERK2], reaching a maximum within 2.5 min when 3.9- and 8.5-fold increases, respectively, were observed. The EGF-inducedincrease of MAPK activity was transient, with only a slight elevationafter 30 min, whereas CCK-stimulated MAPK remained at a high level ofactivation to 60 min. The protein kinase C inhibitor GF-109203Xabolished the activation by phorbol ester and inhibited the effect ofCCK by 78% but had no effect on EGF-activated MAPK activity. EGF and CCK activated both forms of MAPK kinase (MEK), with CCK having a muchlarger effect, activating MEK1 by 6-fold and MEK2 by 10-fold, whereasEGF activated both MEKs by only 2-fold. Immunoblotting revealed threedifferent forms of Raf in pancreatic acinar cells. Of the total basalRaf kinase activity, 3.7% was Raf-A, 89.0% was Raf-B, and 7.3% wasc-Raf-1. All three forms of Raf were stimulated to a greater extent byCCK than by EGF, which was especially evident for Raf-A and c-Raf-1.The effect of CCK in activating Rafs was at least partially mimicked bystimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. EGF significantlyincreased GTP-bound Ras by 183 and 164% at 2.5 and 10 min,respectively; CCK and TPA had no measurable effect. Our study suggeststhat CCK and EGF activate the MAPK cascade by distinct mechanisms in pancreatic acinar cells.

     

Alpha 1-adrenergic partial agonists utilize cytosolic Ca2+ more effectively for contraction in aortic smooth muscle

Fura 2 loaded thoracic aorta strips from rabbits were used. Norepinephrine, phenylephrine, clonidine, and tizanidine induced an increase in cytosolic Ca2+ concentration [( Ca2+]i) and muscle tension in a concentration-dependent manner. A positive correlation between [Ca2+]i and tension development owing to the agonists was noted. The slope of regression lines between [Ca2+]i and tension development for clonidine and tizanidine, alpha 1-adrenergic partial agonists, were significantly steeper than those for norepinephrine and phenylphrine, alpha 1-adrenergic full agonists. The intrinsic activities of the partial agonists obtained from tension development were greater than those from changes in [Ca2+]i. These results suggest that the partial agonists cause a greater muscle tension than the full agonists at the same level of [Ca2+]i.     

Somatostatin and alpha 2-adrenergic agonists selectively inhibit vasopressin-induced cyclic AMP accumulation in MDCK cells

The effect of somatostatin and alpha 2-adrenergic agonists on cyclic AMP accumulation was examined in MDCK cells, grown in defined medium. These hormones inhibited vasopressin-induced cyclic AMP formation, without affecting either the basal or the glucagon- and prostaglandin E2-stimulated level. Pretreating the cells with pertussis toxin, or incubating them with MnCl2 at a low concentration reversed the effect of somatostatin and alpha 2-agonists. These results suggest that somatostatin and norepinephrine could selectively modulate the renal effect of vasopressin, via the inhibitory regulatory subunit (Ni) of adenylate cyclase.     

Changes in nerve terminals and acini of the lacrimal gland and changes in secretion induced by autonomic denervation

Summary Nerve terminal alterations induced by superior cervical ganglionectomy and pterygopalatine ganglion lesions, and acinus cell alterations induced by the latter operation and by greater petrosal neurectomy, established that a majority of interstitial and all parenchymal nerve terminals of the lacrimal gland in monkeys were parasympathetic. These were shown to originate from neurones of the pterygopalatine ganglion. A minority of interstitial terminals were sympathetic and were distinguished from the remainder by the presence of small granular vesicles. The number of small granular vesicles was increased by iproniazid treatment.The production of serous but not of mucous secretion granules was shown to be dependent upon a functioning parasympathetic nerve supply. Lacrimal secretion was not appreciably altered by sympathectomy but was radically reduced by parasympathectomy.Nerve terminals were closely apposed to between 15 and 56% of capillary sections. Both parasympathetic and sympathetic terminals innervated capillaries.The various experimental results are used to postulate a peripheral control mechanism for lacrimal gland secretion involving only parasympathetic nerve terminals.The author expresses his appreciation to Professor R. Warwick for the use of facilities of the Anatomy Department, Guy's Hospital Medical School.     

Serotonin regulates the secretion and autocrine action of a neuropeptide to activate MAPK required for long-term facilitation in Aplysia

In Aplysia, long-term facilitation (LTF) of sensory neuron synapses requires activation of both protein kinase A (PKA) and mitogen-activated protein kinase (MAPK). We find that 5-HT through activation of PKA regulates secretion of the sensory neuron-specific neuropeptide sensorin, which binds autoreceptors to activate MAPK. Anti-sensorin antibody blocked LTF and MAPK activation produced by 5-HT and LTF produced by medium containing sensorin that was secreted from sensory neurons after 5-HT treatment. A single application of 5-HT followed by a 2 hr incubation with sensorin produced protein synthesis-dependent LTF, growth of new presynaptic varicosities, and activation of MAPK and its translocation into sensory neuron nuclei. Inhibiting PKA during 5-HT applications and inhibiting receptor tyrosine kinase or MAPK during sensorin application blocked both LTF and MAPK activation and translocation. Thus, long-term synaptic plasticity is produced when stimuli activate kinases in a specific sequence by regulating the secretion and autocrine action of a neuropeptide.     

Comparative effects of cytochalasin D on the protein discharge induced by alpha- and beta-adrenergic or cholinergic agonists in rat exorbital lacrimal glands

Cytochalasin D altered the kinetics of peroxidase and radiolabeled protein discharge from rat exorbital lacrimal glands in vitro, in response to various secretagogues. The changes were different with each inducer. The discharge due to isoproterenol was immediately inhibited by 95%; the discharge evoked by noradrenaline via alpha-adrenergic receptors was progressively reduced and was inhibited by 50% after 30 min, whereas that evoked by carbachol was not influenced during the initial discharge period and was diminished by only 30% after 30 min. When calcium was removed from the incubation medium, the secretory responses were lowered and the inhibitory effect of cytochalasin D was still observed. The rate of protein discharge inhibition was related to the dose and was maximal with 2 X 10(-6) M cytochalasin D when the discharge resulted from cholinergic, alpha- or beta-adrenergic or dibutyryl cAMP stimulation. Cytochalasin D did not impair cellular energetics nor other stimulations induced through muscarinic or adrenergic receptors. Cytochalasin D effects could be related to interaction with actin, leading to the inhibition of the release of proteins into the incubation medium following the activation of the adrenergic system.     

Introduction of BAPTA into intact rat submandibular acini inhibits mucin secretion in response to cholinergic and beta-adrenergic agonists

Incorporation of the calcium chelator BAPTA into isolated, intact rat submandibular acini by hypotonic swelling, resulted in complete inhibition of carbamylcholine, noradrenaline and isoproterenol stimulation of mucin secretion. No effects of intracellular BAPTA on cell viability or beta-adrenergic stimulation of cyclic AMP formation were observed. The data are the first to demonstrate that calcium is necessary for beta-adrenergic stimulation of secretion and suggest that Ca2+ provides a common link in the triggering of exocytosis.