Does the angle of trocar insertion affect the fascial defect caused? A porcine model.

INTRODUCTION: With an incidence of 0-5.2%, trocar site hernias frequently occur following laparoscopy. It is unclear to what extent the angle of trocar insertion affects the size of the fascial defect caused. Hence, we performed a porcine model. METHODS: In October 2022, a total of five female pigs were euthanized. In alternating order, three bladeless and two bladed conical 12-mm trocars were inserted at an angle of 45° on each side for 60 min twice each pig. For this purpose, an epoxy resin handmade cuboid with a central channel that runs at an angle of 45° was used. Subsequently, photo imaging and defect size measurement took place. The results were compared with those of our previously conducted and published porcine model, in which the trocars were inserted at an angle of 90°. Effects of trocar type (bladed vs. bladeless) and angle on defect size were analyzed using a mixed model regression analysis. RESULTS: The bladeless trocars caused statistically significant smaller defects at the fascia than the bladed (23.4 (SD = 16.9) mm2 vs. 41.3 (SD = 14.8) mm2, p < 0.001). The bladeless VersaOne trocar caused the smallest defect of 16.0 (SD = 6.1) mm2. The bladed VersaOne trocar caused the largest defect of 47.7 (SD = 10.5) mm2. The defect size of the trocars used at a 45° angle averaged 30.5 (SD = 18.3) mm2. The defect size of trocars used at a 90° angle was significantly larger, averaging 58.3 (SD = 20.2) mm2 (p = 0.007). CONCLUSION: When conical 12-mm trocars are inserted at a 45° angle, especially bladeless ones, they appear to cause small fascial defects compared with insertion at a 90° angle. This might lead also to a lower rate of trocar hernias. Bladeless trocars might cause smaller fascial defects than bladed trocars.

How often is prophylactic parastomal mesh placement performed after rectal resection without sphincter preservation? An analysis of German nationwide hospital discharge data among 41,697 patients.

PURPOSE: The European Hernia Society guidelines of parastomal hernias, published in 2017, strongly recommend prophylactic synthetic non-absorbable mesh upon the construction of a permanent end colostomy to reduce the incidence of parastomal hernias. This study aims to evaluate the implementation of the guidelines in Germany. METHODS: This is a retrospective multicentric analysis conducted in December 2022 at the University Hospital Brandenburg an der Havel. Anonymous data on rectal resection without sphincter preservation in the period 2010-2020 were extracted from the German nationwide hospital discharge data set. Individuals with a hernia and < 18 years old were excluded. Another exclusion criterion was a performed colectomy or proctocolectomy with an ileoanal pouch and placement of an absorbable mesh. The primary endpoint was the annual rate of prophylactic parastomal mesh placement following rectal resection without sphincter preservation in Germany. Cases reporting both non-absorbable mesh placement and rectal resection without sphincter preservation were considered prophylactic mesh insertions. RESULTS: A total of 41,697 patients received a rectal resection without sphincter preservation and without non-absorbable mesh placement. Among these individuals, 27,089 were male and 14,608 were female. The rate of reoperations (3.1%) and the length of hospital stay (25.3 days ± 19.32) remained almost constant during these 10 years. The rate of prophylactic mesh placement was increasing from 0.2% (n = 8) in 2010 to 6.4% (n = 198) in 2020. CONCLUSIONS: Currently, only the minority of patients who have undergone rectal resection without sphincter preservation receive prophylactic mesh insertion.

Mechanical responses of tracheal tissue in vitro: dependence on the tissue preparation employed and relationship to smooth muscle content.

We examined the relationship between the quantity of smooth muscle in isolated tracheal preparations and their responses to contractile agonists. The responses of two different tracheal preparations, rings and tubes, to carbachol and serotonin were compared both intra-species (Fisher vs. Lewis strain rats) and inter-species (rat vs. guinea-pig). The rank order for carbachol-induced maximal isometric tensions was Fisher > Lewis > guinea-pig and for serotonin Fisher > guinea-pig > Lewis for tracheal rings. The sensitivities to carbachol and serotonin were greater in Fisher than in Lewis rats. Guinea-pig tracheal rings were comparable to Fisher in sensitivity to carbachol, but were more sensitive to serotonin than either Fisher or Lewis rings. In both species, agonist-independent differences were found in the maximal tension of rings taken from different regions of trachea. For whole tracheal tubes under isovolumetric conditions, the rank order for carbachol-induced changes in the intraluminal pressure was guinea-pig > Lewis > or = Fisher. The sensitivity to carbachol was greater in guinea-pig tubes than in rat. The quantity of tracheal smooth muscle estimated from myosin was greater in guinea-pigs than in either Fisher or Lewis rats. In addition, the area of cartilage determined by morphometry in guinea-pig trachea was greater than that in the rat. We conclude that a concordance between the magnitude of contraction and the amount of tracheal smooth muscle is obtained only in whole tracheal tubes and not in tracheal rings. Several factors could contribute to the observed discrepancies in tracheal rings, including regional differences in efficacy and sensitivity to contractile agonists and the thickness of cartilage.

Airways of a hyperresponsive rat strain show decreased relaxant responses to sodium nitroprusside.

The aim of the current studies was to investigate the possibility that a decreased relaxant response to nitric oxide (NO) might contribute to strain-related differences in airway responsiveness in the rat. Isolated tracheal rings from hyperresponsive. Fisher rats were confirmed to be more responsive to carbachol [mean effective concentration (EC50) = 2.45 x 10(-7) M] than those from Lewis (EC50 = 3.60 x 10(-7) M, P < 0.03) and ACI (EC50 = 3.85 x 10(-7) M, P < 0.01) rats. Sodium nitroprusside (SNP), a NO donor, caused relaxation of the carbachol (10(-6) M) contracted tracheal rings, but the half-maximal inhibition concentration (IC50) SNP in Fisher rats (5.60 x 10(-6) M) was significantly higher than in Lewis (1.34 x 10(-6) M, P < 0.001) and ACI rats (1.13 x 10(-6) M, P < 0.0005). The inhibitory effect of SNP on airway responsiveness to inhaled methacholine (MCh) in vivo was also less pronounced for Fisher than Lewis rats. SNP induced an accumulation of guanosine 3',5'-cyclic monophosphate (cGMP) in cultured tracheal smooth muscle cells (TSM). Fisher TSM produced less cGMP on exposure to SNP compared with TSM from ACI (P < 0.01) and Lewis (P < 0.0001) rats. A decreased guanylyl cyclase activity may account for the impaired relaxant effect of SNP in Fisher rats.

Bronchoalveolar lavage fluid from immature rats with hyperoxia-induced airway remodeling is mitogenic for airway smooth muscle.

We previously demonstrated that hyperoxia-exposed immature rats develop airway smooth muscle layer thickening; this remodeling appears partially attributable to smooth muscle hyperplasia. In this study, we tested the hypothesis that excess mitogenic activity for airway smooth muscle cells is present within the lungs of hyperoxia-exposed immature rats. We assessed the proliferative effect of bronchoalveolar lavage (BAL) fluid from air- and O2-exposed animals on cultured rat tracheal smooth muscle cells. BAL fluids from air- or O2-exposed immature rats increased DNA synthesis ([3H]-thymidine incorporation at 24 h of incubation) and cell number (compared with DMEM-treated control cells, at 2 days of incubation), but BAL fluid from O2-exposed animals had significantly greater mitogenic effects. This excess mitogenic activity was lipid inextractable and was ablated by trypsin digestion, indicating that at least one polypeptide growth factor was responsible; molecular sieve fractionation demonstrated a molecular weight of > 10 kD. Because platelet-derived growth factor (PDGF) has been identified in other models of hyperoxia exposure, we tested the further hypothesis that PDGF contributes to the observed excess mitogenic activity. Addition of neutralizing anti-PDGF antibodies to BAL-stimulated smooth muscle cultures did not reduce BAL fluid-induced mitogenesis. These data indicate that the lungs of O2-exposed rats contain excess mitogenic activity for airway smooth muscle, attributable to non-PDGF polypeptide growth factors. It is conceivable that this abnormal mitogenic activity contributes to O2-induced airway smooth muscle remodeling observed in immature rats in vivo.

Antiproliferative effect of prostaglandin E2 in cultured guinea pig tracheal smooth muscle cells.

The respiratory epithelium plays an important role in modulating airway smooth muscle responsiveness to contractile agonists, and damage of the epithelium may be involved in the pathogenesis of bronchial hyperresponsiveness. This study was undertaken to determine whether prostaglandin E2 (PGE2), a relaxant agent synthesized and released by airway epithelial cells, could exert long-term effects on airway smooth muscle by regulating cell proliferation. Incubation of growth-arrested tracheal smooth muscle cells with serum for 24 h stimulated DNA synthesis in a concentration-dependent manner, as determined by [3H]thymidine incorporation. PGE2 and forskolin, a direct activator of adenylate cyclase, inhibited serum-induced cell proliferation, and the effects were dose dependent. PGE2 and forskolin also stimulated adenosine 3',5'-cyclic monophosphate accumulation. An inhibitor of cyclooxygenase, indomethacin, enhanced DNA synthesis induced by serum. These results indicate that exogenous PGE2 exerts an antiproliferative effect on smooth muscle cells in culture by activation of adenylate cyclase and suggest a role for the epithelium in modulating airway smooth muscle growth.

P2-purinergic receptors in human breast tumor cells: coupling of intracellular calcium signaling to anion secretion.

ATP increases intracellular Ca++ ([Ca++]i) by activating different P2-purinoreceptors. Because ATP increases Cl- secretion in cystic fibrosis (CF)-affected epithelia, the current study was designed to establish the link between these two events. Studies were done in epithelial, human MCF-7 breast tumor cells in which the presence of mRNA transcripts encoding CF transmembrane conductance regulator was initially established. Changes in [Ca++]i were measured in single cells by fluorescence microscopy; anion transport was measured by 125I efflux. ATP stimulated concentration-dependent increases in [Ca++]i and 125I efflux from MCF-7 cells. The relative order of agonist potency of various selective P2-purinoreceptor agonists in increasing [Ca++]i and 125I efflux was: UTP > or = ATP > ADP = AMP; 2-chloro-ATP, 2-methylthio-ATP and AMP-phencyclidine were considerably less potent than ATP. The Ca++ ionophore ionomycin increased both intracellular [Ca++]i and 125I secretion. Exposing cells to the intracellular chelator ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetra-acetic acid (EGTA)-acetoxymethylester decreased (AM) decreased ATP- and ionomycin-stimulated 125I efflux. Extracellular EGTA did not alter the Ca++ response to ATP, but inhibited the response to ionomycin. The chelator inhibited both ATP- and ionomycin-induced 125I secretion. Exposure of cells to nifedipine did not affect the responsiveness of MCF-7 cells to ATP. The anion transport antagonist 4,4'-diisothiocyananatostilbene-2,2'-disulfonic acid partially inhibited ATP- and cationophore-stimulated increases in [Ca++]i and 125I secretion. The data suggest that activation of P2 receptors in MCF-7 cells leads to an increase in anion transport as a result of the ability of ATP to increase [Ca++]i; moreover, anion channel antagonists may produce their inhibitory effect on 125I secretion, in part, by blocking agonist-induced intracellular Ca++ signaling.

Diphenylamine-2-carboxylate analogues block Cl- conductances in A7r5 cells by affecting cellular Ca2+ homeostasis.

We have investigated the cellular signalling pathway by which vasopressin stimulates a Ca2(+)-dependent Cl- conductance and the effects of two known Cl- channel blockers in cultured rat A7r5 aortic smooth muscle cells using anion efflux and fluorescent Ca2+ imaging studies. Addition of vasopressin (100 nM) to A7r5 cells enhanced 125I (Cl- substitute) efflux from the cells through a V1 receptor-mediated pathway. Maximal increases in the rate of efflux were observed 1 min following addition of vasopressin (4-fold above basal levels). Activation of the V1 pathway was demonstrated by an increase in inositol trisphosphate (IP3) formation and lack of cAMP accumulation by the cells following the addition of vasopressin. Fluorescent ratio imaging with fura-2 revealed that addition of vasopressin to the cells results in an increase of [Ca2+]i which peaks within 20 s and does not return to resting levels during the 100 s observation period. The addition of a Ca2+ ionophore mimicked the vasopressin-induced efflux from the cells. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and a chloro-substituted compound (cpd 149) inhibited the vasopressin-stimulated 125I efflux from the cells. The concentrations of NPPB and cpd 149 required to inhibit 125I efflux from the cells were similar to those which also attenuated vasopressin-induced Ca2+ transients in the cells. NPPB and cpd 149 had no effects on the ionomycin stimulated efflux. The mechanism(s) by which cpd 149 exerts its effect on stimulated efflux was examined by measuring its action on vasopressin-induced changes in IP3. Compound 149 inhibited IP3 generation in response to vasopressin.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of adenylate cyclase-coupled histamine H2 receptors in guinea pig tracheal smooth muscle cells in culture and the effect of dexamethasone.

Histamine acts on airway contractile elements through at least two different receptor subtypes: H1, which mediates Ca(2+)-dependent contraction, and H2, which stimulates cyclic adenosine monophosphate (cAMP) synthesis and possibly relaxation. The aim of this study was to determine the relative contribution of the different receptor subtypes to histamine-stimulated cAMP production by guinea pig tracheal smooth muscle (GPTSM) cells in primary culture. Histamine and N-alpha-methylhistamine induced concentration-dependent cAMP synthesis; these effects were entirely blocked by 10(-4) M cimetidine, an H2-receptor antagonist, whereas 10(-6) M thioperamide, a selective H3 blocker, was ineffective. The H3 agonist, R-(alpha)-methylhistamine, did not stimulate cAMP synthesis. Triprolidine, an H1 antagonist, did not modify histamine (10(-5) M)-stimulated cAMP synthesis. Histamine (10(-5) M) doubled [Ca2+]i in GPTSM. A 24-h pretreatment of GPTSM cells with 10(-6) M dexamethasone enhanced cAMP synthesized in response to 10(-5) M histamine and to 5 x 10(-6) M forskolin but did not significantly alter either the affinity or the binding capacity for [3H]-tiotidine, an H2-receptor antagonist. These results indicate that GPTSM cells in culture express H2 but not H3 receptors, which are linked to adenylate cyclase; their functional expression does not seem to be modulated by the concurrent activation of H1 receptors, whose presence in GPTSM is evidenced by a histamine-stimulated increase in [Ca2+]i. The most likely site of action of dexamethasone in enhancing histamine-stimulated cAMP synthesis is at the level of adenylate cyclase since the steroid had no effect on the H2 receptor itself.

Extracellular ATP stimulates elastase secretion from human neutrophils and increases lung resistance and secretion from normal rat airways after intratracheal instillation.

The effect of ATP on intracellular Ca2+ levels and elastase secretion in isolated normal human peripheral blood neutrophils was investigated as was its in vivo effect on lung resistance and mucous secretion. ATP (10(-5) M) increased [Ca2+]i from 61 +/- 3 to 165 +/- 15 nM in nonactivated neutrophils; elastase secretion was increased by 40% from nonactivated neutrophils but was unaffected in fMLP (10(-5) M) activated cells. Instillation of ATP (10(-5) and 10(-3) M) into the airways of brown Norway rats increased both lung resistance and secretion. These findings suggest that aerosolization of ATP into the cystic fibrosis-affected bronchial tree might be hazardous in terms of enhancement of parenchymal damage, which would result from neutrophil elastase release, and in terms of impaired respiratory lung function.

ACTH secretion from mouse pituitary tumor cells is inhibited by loop diuretic drugs.

Stilbene disulfonate and phenylanthranilic acid derivatives block Cl- transport and ACTH secretion from mouse AtT-20 clonal corticotrophs. This study was to determine whether antagonism of Na+/K+/Cl- co-transport by loop diuretics (furosemide or bumetanide) and thereby Cl- entry could block ACTH secretion. Activity of the Na+/K+/Cl- co-transporter (symport) was measured as ouabain-insensitive, loop diuretic-sensitive uptake of 86Rb. Ouabain-insensitive uptake (80% of total 86Rb uptake) was linear over 10 min and was markedly reduced by furosemide. Substitution of Na+ by choline or Cl- by gluconate resulted in an 82 and 55% decrease, respectively, in 86Rb uptake. Furosemide and bumetanide incompletely inhibited 86Rb uptake (maximal inhibition of 60% and 69%, respectively; IC50: 1.6 x 10(-5) and 3 x 10(-6) M, respectively). Forskolin did not affect the activity of the Na+/K+/Cl- co-transporter but both basal and forskolin-stimulated secretion of ACTH were inhibited by furosemide (IC50 of 5 x 10(-4) M; maximal inhibition: 50%). Bumetanide did not affect forskolin-induced cAMP synthesis. Use of cyclamate or gluconate in place of Cl- also resulted in the inhibition of basal and forskolin-stimulated ACTH secretion. The data support the belief that inhibition of Cl- entry into AtT-20 cells by way of an Na+/K+/Cl- co-transporter can result in the inhibition of ACTH secretion but that other anion entry mechanisms may also be coupled to the secretory response in these cells.

Antagonists of epithelial chloride channels inhibit cAMP synthesis.

In past studies we observed that the chloride channel blocker, diphenylamine-2-carboxylate (DPC) and chemically related drugs (Hoechst compounds 131, 143, 144) inhibited cAMP formation in mouse pituitary tumor cells. The object of this study was to determine whether these drugs inhibited chloride transport in human T-84 colonic carcinoma cells through an effect on cAMP metabolism. Chloride secretion (measured as 125I efflux from isotope-preloaded cells) was stimulated in a concentration-dependent manner by vasoactive intestinal polypeptide (VIP) (EC50 = 1.5 x 10(-10) M) which similarly increased cAMP synthesis (EC50 = 1.6 x 10(-8) M). The cAMP response to VIP was inhibited 17, 52, 55, and 78% maximally by DPC and compounds 144, 143, and 131, respectively. In untreated T-84 cells, 125I secretion fell by 66% after 3 min; VIP (10(-7) M) increased secretion about fivefold over the same period. Both basal and VIP-stimulated 125I secretion were inhibited up to 60% by compound 131. Pretreatment of cells with pertussis toxin did not attenuate the inhibitory effect of channel blockers on either VIP-stimulated cAMP synthesis or 125I secretion. The cationophore, A-23187, which had no effect on cAMP formation, and 8-Br-cAMP both stimulated 125I secretion from T-84 cells. These secretory responses were inhibited by compound 131. The mechanism by which phenylanthranilic acids antagonize cAMP synthesis and its significance is not known; however, the data suggest that this family of drugs may inhibit chloride transport by both cAMP-dependent and independent mechanisms.

Chloride channel blockers inhibit ACTH secretion from mouse pituitary tumor cells.

The effect of several chemically related chloride channel blocking drugs was investigated on the adrenocorticotropic hormone (ACTH) secretory process in mouse clonal AtT-20 corticotrophs. When cells were simultaneously exposed to diphenylamine-2-carboxylate (DPC) or related substances (Hoechst compounds 131, 143, and 144) and the adenylate cyclase activator forskolin, ACTH secretion was inhibited by 76-95% [half-maximal inhibitory concentration (IC50) 450, 15, 84, and 32 microM, respectively]. All four compounds also blocked forskolin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) synthesis in AtT-20 cells by 51-87% (IC50 190, 29, 100, and 130 microM for DPC and compounds 131, 143, and 144, respectively). Pertussis toxin pretreatment of cells caused a partial reversal of DPC-inhibited forskolin-stimulated cAMP formation. The toxin had no effect on inhibition of forskolin-stimulated ACTH secretion by DPC. Secretion of ACTH in response to cAMP-independent stimulants such as the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate or the calcium channel agonist BAY K 8644 were blocked by compound 131 as was the secretory response to 8-bromoadenosine 3',5'-cyclic monophosphate. These results suggest that phenylanthranilic acids have adenylate cyclase inhibiting action but that the postcyclase activity is more relevant to the ability of these compounds to block ACTH secretion. DPC also blocked 125I efflux (an index of Cl- secretion) from AtT-20 cells. Because an increase in osmotic strength of the culture media reduced forskolin-stimulated ACTH secretion, these data suggest that DPC and related compounds may negatively modulate chloride-dependent osmotically driven ACTH secretion from AtT-20 cells.

Atrial natriuretic peptide stimulates submandibular gland synthesis and secretion of cGMP.

Binding of atrial natriuretic peptide (ANP) to rat submandibular gland and its effect on guanosine 3',5'-cyclic monophosphate (cGMP) formation and salivary secretion were investigated. Membranes rapidly and specifically bound 125I-ANP. Binding was inhibited by unlabeled ANP (IC50 approximately 1.6 nM), but not by atriopeptin I, other COOH- and NH2-terminal deleted ANP fragments, or agents such as pilocarpine or substance P. Scatchard analysis revealed a single class of high-affinity sites (dissociation constant 0.74 +/- 0.25 nM; maximal binding capacity 20.5 +/- 6.3 pmol/mg protein). Intravenous infusion of ANP with pilocarpine caused a significant dose-dependent increase in the levels of cGMP detected in plasma and saliva. Because salivary cGMP may have originated in plasma, the effect of ANP on cGMP formation was evaluated in dispersed cells. ANP evoked a concentration-dependent increase in both cGMP synthesis and secretion (EC50 approximately 1.7 x 10(-8) M). The atrial peptide did affect basal or l-isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate synthesis in dispersed cells. When infused by itself and/or with pilocarpine, ANP did not alter the rate of spontaneous or pilocarpine-induced salivary flow, secretion of chloride, or protein release. The data demonstrate the presence of guanylate cyclase-coupled ANP receptors in submandibular gland; the atrial peptide, however, does not exert an effect of the secretory function of the gland.

Y-1 adrenocortical tumor cells contain atrial natriuretic peptide receptors which regulate cyclic nucleotide metabolism and steroidogenesis.

The effects of atrial natriuretic peptide (ANP) on adrenocortical fasciculata cells were examined in the ACTH-responsive Y-1 mouse adrenocortical tumor cell line. Y-1 cell membranes rapidly bound [125I]ANP, with equilibrium binding (22 C) reached within 45 min. Binding of [125I]ANP was inhibited in a concentration-dependent manner by unlabeled ANP and atriopeptin-I (IC50, approximately 1.2 X 10(-9) and 1.6 X 10(-8) M, respectively), but not by C- or N-terminal-deleted ANP fragments, ACTH, or arginine vasopressin (up to 10(-6) M). Scatchard analysis revealed a single class of high affinity binding sites with a Kd of 1.6 X 10(-10) M and a binding capacity of 560 fmol/mg protein. Photo-affinity labeling demonstrated the specific binding of ANP to two protein entities of 130 and 63 kDa. ANP stimulated both cGMP synthesis and secretion from Y-1 cells (EC50, approximately 3.5 X 10(-9) M). Release of the nucleotide was inhibited by probenecid (IC50, approximately 5 X 10(-5) M). The atrial peptide partially inhibited ACTH-stimulated cAMP formation (IC50, approximately 10(-8) M) and partially antagonized basal and ACTH-stimulated steroidogenesis. The data demonstrate the presence in Y-1 cells of specific and saturable ANP receptors, activation of which leads to changes in cyclic nucleotide metabolism and inhibition of steroidogenesis.

4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid inhibits adrenocorticotropin secretion from anterior pituitary cells.

Mouse clonal ACTH-secreting corticotrophs (AtT-20 cells) possess a membrane Ca2+-activated Cl- conductance which is partially blocked by the disulfonic stilbene derivative 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). In the current study the effect of SITS on the ACTH secretory process was evaluated. SITS markedly blocked basal and forskolin-stimulated ACTH secretion from AtT-20 cells (IC50 = 2.7 x 10(-4) M). Both CRF-induced ACTH secretion and forskolin-stimulated GH secretion from acutely dispersed rat anterior pituitary cells were inhibited by SITS (IC50 = 2.4 and 1.3 x 10(-4) M, respectively). SITS did not alter unstimulated or forskolin-elicited cAMP synthesis in AtT-20 cells, and in fact, could inhibit ACTH secretion in response to cAMP-independent agonists such as the calcium channel activator BAY-K-8644 or the protein kinase-C activator 12-tetradecanoyl-phorbol-13-acetate (IC50 = 2.6 and 2.4 x 10(-4) M, respectively). SITS did not alter the secretion of amylase from isolated exocrine pancreatic acinar cells. Its action was also fully reversible; after its removal from the incubation medium, cells secreted ACTH without a change in response to forskolin activation. Increasing extracellular Ca2+ or the addition of up to 10(-3) M tetraethylammonium or 4-aminopyridine did not reverse the inhibitory pattern of SITS action, suggesting that its inhibitory effect is most likely not due to hyperpolarization of AtT-20 cell membranes. The inability of amiloride to inhibit ACTH secretion further suggests that inhibition of ACTH secretion provoked by SITS is not due to a blockade of Cl-/HCO3- exchange. On the other hand, SITS was able to block 44% of basal 36Cl uptake by AtT-20 cells. Exchange of incubation medium chloride for gluconate or a reduction in the osmotic strength of the medium reduced both basal and secretagogue-stimulated ACTH secretion. The data suggest that SITS may modulate chloride-dependent, osmotically driven secretion from AtT-20 cells.

Phospholipase A2 activation by melittin causes amylase release from exocrine pancreas.

Phospholipase A2-induced deacylation of membrane phospholipids is associated with changes in membrane fluidity. The importance of this reaction in the pancreatic amylase secretory process was tested using melittin, a phospholipase A2 stimulating peptide. Phospholipase A2 activity (using [3H]arachidonic acid release as an index) and amylase secretion were both increased in a time- and concentration-dependent manner by melittin. Phospholipids prelabelled with [3H]oleic acid or [14C]linoleic acid also released radioactive free fatty acids in response to melittin. Prostaglandin synthesis was not involved in the melittin response, since inhibitors of arachidonic acid oxidation (indomethacin, 5,8,11,14-eicosatetraynoic acid) did not alter the ability of melittin to release [3H]arachidonic acid or amylase. When melittin was co-applied with carbachol, cholecystokinin octapeptide, or vasoactive intestinal peptide, amylase secretion was additive. The effect of melittin on both fatty acid and amylase release was dependent on extracellular calcium, though melittin's effects were not dependent on the intracellular accumulation of second messengers such as calcium or cAMP. The data suggest that activation of phospholipase A2 by melittin results in the triggering of the secretory process in exocrine pancreas by a different mechanism than that for other pancreatic secretagogues.

Is atrial natriuretic peptide synthesized and internalized by gonadotrophs?

The present study was designed to determine whether atrial natriuretic peptide (ANP) could be both synthesized and internalized by rat anterior pituitary gonadotrophs. ANP synthesis was assessed by in situ hybridization of ultrathin frozen sections of anterior pituitary to a biotinylated 30-base oligonucleotide to rat ANP mRNA. As revealed by the immunogold technique, only gonadotrophs were labeled by the probe. At the subcellular level, ANP mRNA was observed at both the nuclear and cytoplasmic levels in gonadotrophs, and labeling of the latter compartment was quantitatively more intense. Internalization of ANP was investigated by an in vivo ultrastructural autoradiographic approach. Intravenous injection of [125I]ANP resulted in rapid labeling within 1 min of the plasma membrane, cytoplasmic matrix, secretory vesicle, and mitochondrial compartments and the Golgi apparatus; these compartments were labeled throughout the remainder of the time course studied (1-30 min). Peak labeling of the plasma membrane compartment was at 1 min and diminished from that point; labeling in the Golgi apparatus peaked 5 min postinjection, while in the other compartments labeling was fairly uniform over the time course. The lysosomal compartment was also radiolabeled; however, only 2 and 5 min after injection of [125I]ANP. The findings demonstrate that gonadotrophs can both synthesize and internalize extracellular ANP. These observations can be extended to suggest that ANP has both autocrine and paracrine actions in the anterior pituitary gland. Since the peptide neither stimulates nor antagonizes the release of any anterior pituitary hormone, these actions are probably unrelated to the adenohypophyseal secretory function.

Direct binding of atrial natriuretic factor to adrenocortical mitochondria.

Atrial natriuretic factor (ANF) is a known antagonist of adrenocortical aldosterone synthesis and secretion. Immunocytochemical and ultrastructural autoradiographic evidence suggests that ANF may bind to mitochondria of a number of target tissues including adrenal cortex. Consequently, the ability of [125I]ANF to bind directly to isolated bovine adrenocortical mitochondria was assessed. Mitochondrial-enriched subfractions of adrenocortical homogenates were prepared by differential and sucrose gradient centrifugation. Mitochondrial membranes specifically bound [125I]ANF. At 20 degrees C equilibrium was achieved between 90 and 120 min. [125I]ANF binding was inhibited in a concentration-dependent manner by unlabelled ANF (IC50 about 5 x 10(-10) M); other substances with biological actions on glomerulosa cells (arginine vasopressin, angiotensin II) did not alter [125I]ANF binding. Similarly shorter ANF fragments including ANF-(103-125), ANF-(99-109) and ANF-(111-126) had no significant competitive effect on binding of the labelled ligand. While Ca2+ and Mg2+ had little effect on ANF binding, the divalent cation Ni2+ inhibited binding of radiolabelled ANF by 90% (IC50 about 8.3 x 10(-5) M). Scatchard analysis revealed both high and low affinity binding sites for [125I]ANF with respective KDs of 4.7 +/- 7 pM and 0.3 +/- 0.02 nM and receptor densities of 1.1 +/- 0.2 and 8.6 +/- 0.1 pmol/mg protein. At 0.2 nM, Ni2+ caused a 5-fold and 100-fold decrease in high and low affinity [125I]ANF binding, respectively. The data demonstrate that ANF binds directly to mitochondria and perhaps it is at this site that the atrial peptide negatively modulates agonist-induced aldosterone biosynthesis.

Immunocytochemical localization, binding, and effects of atrial natriuretic peptide in rat adipocytes.

The metabolic effects of atrial natriuretic peptide (ANP) have not been widely investigated. Since adipocyte cells represent a model system extensively used to examine the metabolic actions of many peptide hormones, we sought to establish whether ANP could bind to adipocyte membranes, alter cyclic nucleotide metabolism, and affect spontaneous or hormone-stimulated lipolysis. Using in vitro autoradiographic techniques, radiolabelled ANP was found to bind specifically to mammary gland fat cells. Additionally, endogenous ANP-like immunoreactivity could be localized in the plasma membrane compartment and cytoplasmic matrix of fat cells, but not in fat vacuoles. [125I]ANP bound to single high affinity sites (Kd = 0.72 nM) in fat cell membranes. The binding was rapid (equilibrium within 1 min at 25 degrees C) and specific. The atrial peptide was capable of stimulating a time- and concentration-dependent increase in cGMP accumulation in isolated adipocytes, but had no effect on spontaneous or stimulated [-)-isoproterenol, ACTH, forskolin) cAMP formation. ANP did not alter the increase in glycerol production stimulated by l-epinephrine in isolated fat cells. While i.v. infusion of ANP stimulated a marked increase in circulating levels of cGMP, the atrial peptide did not alter plasma triglyceride levels. These data demonstrate the presence of specific ANP binding sites on adipocyte membranes and internalization of ANP-associated immunoreactivity. These receptors are biochemically functional given the ability of ANP to augment cGMP formation. The peptide, however, does not exert an action on adipocyte lipolysis. Adipocytes, therefore, represent an ANP target tissue in which the physiological action of the peptide is yet to be defined.