Scholarship in teaching: an imperative for the 21st century.

At some medical schools broader definitions of scholarship have emerged along with corresponding changes in their academic reward systems. Such situations are not common, however. The definition of scholarship generally applied by medical schools is unnecessarily narrow and excludes areas of legitimate academic activity and productivity that are vital to the fulfillment of the school's educational mission. The authors maintain that creative teaching with effectiveness that is rigorously substantiated, educational leadership with results that are demonstrable and broadly felt, and educational methods that advance learners' knowledge are consistent with the traditional definition of scholarship. Faculty whose educational activities fulfill the criteria above are scholars and must be recognized by promotion. The authors specifically address scholarship in education, focusing on teaching and other learning-related activities rather than on educational research, which may be assessed and rewarded using the same forms of evidence as basic science or clinical research. They build on Boyer's work, which provides a vocabulary for discussing the assumptions and values that underlie the roles of faculty as academicians. Next, they apply Glassick et al.'s criteria for judging scholarly work to faculty members' educational activities to establish a basis for recognition and reward consistent with those given for other forms of scholarship. Finally, the authors outline the organizational infrastructure needed to support scholars in education.

Morphine tolerance and dependence in the rat intestine in vivo.

There has been no previous demonstration of opioid tolerance and dependence with respect to the propulsive and contractile activities of the gut in vivo. In the experiments described herein, morphine was administered continuously (1 mg/kg/hr s.c., 72 hr) and/or by bolus injection (2 mg/kg) and intestinal motility and transit were evaluated in unanesthetized rats. Tolerance in intestinal motility (contractions) and propulsion (transit) was measured in two ways, i.e., by measuring the time required for motility and propulsion to return to control values and by measuring the loss of effectiveness of bolus morphine administered to animals receiving continuous infusion of the opiate. The dose of morphine chosen for continuous administration (1 mg/kg/hr s.c. via Alzet minipumps) was based on the dose at which morphine inhibited intestinal propulsion by 50%. Morphine (1 mg/kg/hr) decreased the frequency of contractions in, and propulsion along, the small bowel and colon and produced mild antinociception. The frequency of duodenal and colonic contractions returned to normal within 13 to 16 hr. After 24 hr of morphine treatment, the inhibitory effects of bolus doses of morphine on motility and transit were diminished; the effects were eventually lost (48 hr). Similarly, the antinociceptive effects of bolus doses of morphine were diminished by 18 hr and lost by 24 hr. Naloxone (0.1 mg/kg s.c.) given to morphine-tolerant animals (72 hr) resulted in an increase in the frequency and amplitude of contractions in the colon, an increase in the propulsive activity of the small intestine and colon and diarrhea. These results provide direct demonstration of opioid tolerance and dependence of contractile and propulsive activity in the rat intestine in vivo.

Cholecystokinin-induced antinociception is not blocked by CCK-A or CCK-B receptor antagonists.

To determine the relative importance of CCK-A, CCK-B, and opioid receptors in mediating the antinociceptive actions of cholecystokinin, we evaluated the actions of selective agonists and antagonists in the mouse hot plate assay. The agonists used were CCK (1-30 nmol i.c.v.), a CCK-A receptor agonist (SNF9019; 0.3-10 nmol i.c.v.), and a CCK-B receptor agonist (SNF9007; 0.3-10 nmol i.c.v.). The antagonists used were the CCK-A receptor antagonist, L364,718 (12.5 nmol i.c.v.), CCK-B receptor antagonist, L365,260 (2.5-25 nmol i.c.v.), and the nonselective opioid receptor antagonist naloxone (1 mg/kg s.c.). CCK and its receptor-selective analogues, SNF9019 and SNF9007, resulted in antinociception that was blocked by naloxone, but was not antagonized by L364,718 or L365,260. In contrast, in positive control experiments, the inhibitory effects of CCK, SNF9019, and SNF9007 on gastrointestinal propulsion in mice were antagonized by identical i.c.v. doses of L364,718 and L365,260. We conclude that centrally administered CCK produces antinociception in the mouse hot plate assay via opioid receptors, but independent of CCK-A or CCK-B receptors. It is necessary to speculate that other CCK receptors, not antagonized by currently available selective antagonists, may exist.

Inhibition of adenylyl cyclase activity by the cholecystokinin analog SNF 9007 in neuroblastoma x glioma NG108-15 hybrid cells.

The effect of the cholecystokininB (CCKB) receptor-selective cholecystokinin octapeptide (CCK-8) analog SNF 9007 on forskolin-stimulated adenylyl cyclase activity in NG108-15 hybrid cells was measured. The activity of SNF 9007 was compared to the delta opioid agonists D-Pen2-D-Pen5-enkephalin (DPDPE, delta 1 receptor-selective) and Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2, (D-Ala2-deltorphin II, delta 2-receptor-selective) because SNF 9007 binds with moderate affinity to delta opioid receptors. SNF 9007 inhibited forskolin-stimulated adenylyl cyclase activity with efficacy similar to DPDPE. IC50 determinations showed that D-Ala2-deltorphin II was the most potent, followed by DPDPE, then SNF 9007 (IC50 values = 0.013, 0.21 and 4.8 microM, respectively). CCK-8 had no effect on adenylyl cyclase activity. The delta 1 receptor-selective antagonist 7-benzylidenenaltrexone hydrochloride (BNTX, 10 nM) had no effect on the activity of any of these agonists, but the delta 2 receptor-selective antagonist naltriben methanesulfonate (NTB, 10 nM) increased IC50 values of all the agonists. Combinations of BNTX and NTB (10 nM each) increased the D-Ala2-deltorphin II IC50 value 12-fold, the DPDPE IC50 value 18-fold and the SNF 9007 IC50 value 26-fold. The effect of the combined delta antagonists on SNF 9007 activity was different from the effect on DPDPE or D-Ala2-deltorphin II activity. These data suggest that the interaction of the CCK-8 analog SNF 9007 with opioid receptors in NG108-15 hybrid cells is different from the interaction of opioid peptides with these receptors.

Regulation of Raf-1 kinase activity by the 14-3-3 family of proteins.

We have identified the beta (beta) isoform of the 14-3-3 family of proteins as an activator of the Raf-1 protein kinase. 14-3-3 was isolated in a yeast two-hybrid screen for Raf-1 kinase domain binding proteins. Purified bovine brain 14-3-3 interacted specifically with both c-Raf-1 and the isolated Raf-1 kinase domain. Association was sensitive to the activation status of Raf-1; 14-3-3 bound to unactivated Raf-1, but not Raf-1 activated by protein kinase C alpha or Ras and Lck. The significance of these interactions under physiological conditions was demonstrated by co-immunoprecipitation of Raf-1 and 14-3-3 from extracts of quiescent, but not mitogen-stimulated, NIH 3T3 cells. 14-3-3 was not a preferred Raf-1 substrate in vitro and did not significantly affect Raf-1 kinase activity in a purified system. However, in cell-free extracts 14-3-3 acted as a Ras-independent activator of both c-Raf-1 and the Raf-1 kinase domain. The same results were obtained in vivo using transfection assays; 14-3-3 enhanced both c-Raf-1- and Raf-1 kinase domain-stimulated expression of AP-1- and NF-kappa B-dependent reporter genes and accelerated Raf-1 kinase domain-triggered differentiation of PC12 cells. We conclude that 14-3-3 is a latent co-activator bound to unactivated Raf-1 in quiescent cells and mediates mitogen-triggered but Ras-independent regulatory effects aimed directly at the kinase domain.

SNF9007: a novel analgesic that acts simultaneously at delta 1, delta 2 and mu opioid receptors.

Intracerebroventricular administration of the synthetic cholecystokinin analog SNF9007 (Asp-Tyr-D-Phe-Gly-Trp-[NMe]-Nle-Asp-Phe-NH2) produced antinociception in the mouse hot-plate and warm water tail-flick tests. The mechanisms of its analgesic actions were assessed by administering antagonists selective for CCK (cholecystokinin octapeptide, sulfated)-A and CCK-B receptors, as well as specific antagonists for the mu opioid receptor (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, 1 microgram i.c.v.), the delta-1 opioid receptor [D-Ala2-Leu5,Cys6]enkephalin, 4.57 nmol i.c.v., 24 hr pretreatment), the delta-2 opioid receptor (naltrindole benzofuran, 25 pmol i.c.v.) and the kappa opioid receptor (nor-binaltorphimine, 10 mg/kg s.c.). The antinociceptive activity of SNF9007 was not a result of the activation of CCK receptors, as treatment with either CCK-A or CCK-B receptor antagonist was ineffective in blocking SNF9007 antinociception. Nor-binaltorphimine and naltrindole benzofuran were completely ineffective in blocking SNF9007 antinociception when administered alone or in combination. However, co-administration of delta-1 or delta-2 opioid receptor antagonists with the mu opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 resulted in a dramatic reduction in analgesic responses to SNF9007. Furthermore, the co-administration of mu+delta-1 + delta-2 opioid receptor antagonists resulted in an even greater inhibition of SNF9007 antinociception (> 10-fold shift). We conclude that SNF9007 acts simultaneously at brain delta-1, delta-2 and mu opioid receptors to induce antinociceptive effects in mice.

Domain mapping of chicken gizzard caldesmon.

Limited proteolysis, affinity chromatography, and immunoblotting have been used to define the domains of chicken gizzard caldesmon, caldesmon120, that interact with calmodulin, F-actin, and a monoclonal antibody prepared using human platelet caldesmon. Treatment of caldesmon120 with chymotrypsin produces groups of fragments near 100, 80, 60, 38, and 20 kDa. Further digestion produces peptides between 40 and 50 kDa. The 100- and 80-kDa peptides cross-react with the monoclonal antibody; the smaller polypeptides do not. The kinetics of cleavage and the antibody studies indicate that the 38- and 80-kDa fragments are the two major pieces of the 120-kDa protein. The 38-kDa fragment, purified by high performance liquid chromatography, and several of its subfragments at 21 and 25 kDa sediment with F-actin, bind to calmodulin-Sepharose in the presence of Ca2+, and are displaced from F-actin by Ca2+-calmodulin. The 80-kDa fragments did not interact with F-actin or calmodulin. We have tentatively placed the 38-kDa fragment at the C-terminal using polyclonal antibodies selected against a beta-galactosidase-caldesmon120 fusion protein produced by a lambda gt11 lysogen. The 38-, 25-, and 21-kDa fragments cross-react with these antibodies; the 80- and 60-kDa fragments do not. Caldesmon77 from human platelets also cross-reacts with these selected antibodies. The results suggest that interacting calmodulin and F-actin binding sites are localized on a 38-kDa C-terminal fragment of caldesmon. The smallest subfragment of this peptide that binds to both F-actin and calmodulin-Sepharose is about 21 kDa. The monoclonal antibody epitope is tentatively localized near the N-terminal of caldesmon77 and must be within 50 kDa of the N-terminal on caldesmon120.

Prostaglandin E2 inhibition of secretagogue-stimulated [14C]aminopyrine accumulation in rat parietal cells: a model for its mechanism of action.

Prostaglandin E2 (PGE2) differentially inhibited histamine and isoproterenol stimulation of [14C]aminopyrine accumulation in rat parietal cell preparations. Low concentrations of PGE2 decreased the maximum response to isoproterenol whereas higher concentrations increased the EC50 of histamine with only a modest effect on the maximum response. Also, PGE2 potentiated dibutyryl cyclic AMP stimulation of aminopyrine accumulation in either the absence or presence of carbachol. In contrast, PGE2 inhibited potentiation between carbachol and histamine due to its inhibitory effect on histamine and possibly also to an inhibitory effect on cholinergic activity. Islet activating protein prevented the inhibitory actions of PGE2. To account for these results a model is presented based on the recent proposal by Gilman (Cell 36: 577-579, 1984) of an interaction between components of adenylyl cyclase stimulatory and inhibitory guanine nucleotide binding proteins.

Isolation and partial characterization of human platelet vinculin.

A 130,000 Mr protein was isolated from human platelets by sequential DEAE-Sephacel and Sepharose Cl-4B chromatography. Low shear viscometric measurements showed that the enriched protein after DEAE-Sephacel chromatography inhibited actin polymerization. This effect was somewhat greater in the presence of EGTA than in the presence of calcium. Further purification by Sepharose Cl-4B chromatography resulted in a complete loss of this inhibitory effect. Studies with fluorescent actin detected no nucleation or "+" end capping activity in either the DEAE-Sephacel- or Sepharose Cl-4B-purified vinculin. Antibodies raised in mice against the 130,000-mol-wt protein were shown to cross-react with chicken gizzard vinculin and a similar molecular weight protein was detected in WI38 cells and, Madin-Darby canine kidney cells. Lysis experiments with the Madin-Darby canine kidney cells indicated that most of the vinculin was soluble in Triton X-100, although some was found associated with the insoluble cytoskeletal residue. By immunofluorescence, vinculin in WI38 cells was localized to adhesion plaques as described by others. Discrete localization in platelets was also detected and appeared to depend on their state of adhesion and spreading. The results of these experiments suggest that human platelets contain a protein similar to vinculin. It is not clear if platelet vinculin is associated with structures analogous to adhesion plaques found in other cell types. The data indicate that the previously reported effects of nonmuscle vinculins on actin polymerization may be due to a contaminant or contaminants.

Isolated parietal cells: adrenergic response and pharmacology.

Isoproterenol (Iso), epinephrine and norepinephrine each stimulated isolated gastric mucosal parietal cells as shown by an increased accumulation of [14C]aminopyrine (AP), an indirect measure of acid secretion. The beta receptor selective agonists metaproterenol, terbutaline and zinterol stimulated AP accumulation to the same extent as Iso, whereas the beta-1 receptor selective agonist dobutamine was only 20% as effective. The general beta receptor antagonists oxprenolol and dl-propranolol and the beta-2 receptor antagonist H35/25 inhibited Iso-stimulated AP accumulation. Receptor stereoselectivity was shown by the approximately 100-fold difference in potency of the I- and d-isomers of propranolol. The alpha receptor antagonists phentolamine and phenoxybenzamine, the beta-1 receptor antagonists metoprolol and practolol and the muscarinic receptor antagonist atropine were without effect. The histamine H2-receptor antagonist cimetidine inhibited Iso-stimulated AP accumulation an average of 40% at a concentration which inhibits completely histamine-stimulated AP accumulation. The data demonstrate that cells of the rat gastric mucosa have adrenergic beta-2 receptors which when stimulated result in an increase in acid secretion. The results also show that the response is in part mediated indirectly by catecholamine-stimulated release of histamine.

Pirenzepine (LS 519): a weak inhibitor of acid secretion by isolated rat parietal cells.

Carbamylcholine-stimulated potentiation of dibutyryl cyclic AMP-induced acid secretion by isolated rat parietal cells was specifically inhibited by the muscarinic cholinergic antagonist pirenzepine at an IC50 of 1.1 microM. In contrast to the weak inhibition by pirenzepine, the potentiation was inhibited by atropine at an IC50 of 4.6 nM. In vivo, pirenzepine is one-tenth as potent as atropine as an inhibitor of acid secretion. The weak activity of pirenzepine shown in this study suggests that its in vivo inhibitory activity is likely due to an interaction with a site involved in the regulation of gastric acid secretion which has higher-affinity muscarinic receptors for pirenzepine than those associated with parietal cells.

Isolation and enrichment of the gastric chief cells of the rat.

The isolation and enrichment of the gastric chief cells of the rat are described. Ultrastructural examination showed 85% enrichment from a mixed population of mucosal cells following their centrifugation through a discontinuous Percoll gradient. When compared to homogenates of the initial mixed cell population, the enriched chief cell population showed over a three-fold increase in pepsin(ogen) content. Preliminary experiments showed that a combination of the secretagogues histamine and carbamylcholine caused a significant increase in pepsin release from enriched chief cell preparations and a concomitant decrease in their pepsin content as compared to untreated cells. The results obtained in this study indicate the feasibility of employing this procedure for the isolation of gastric chief cells for the in vitro study of secretagogue regulation of pepsin secretion.

Isolated parietal cells: histamine response and pharmacology.

Isolated, partially purified or enriched rat gastric mucosal parietal cells were shown to respond to histamine and other histaminic H2-receptor agonists as measured by an increased accumulation of [14C]aminopyrine. The response was temperature-dependent, related to parietal cell purity and inhibited selectively and reversibly by H2-receptor antagonists. H1-receptor antagonists noncompetitively inhibited histamine, carbamylcholine and dibutyryl cyclic AMP-stimulated aminopyrine accumulation. The affinity constants calculated for the H2-receptor agonists and antagonists were similar to those previously determined in the studies of activation and inhibition of adenylyl cyclase in enriched parietal cell preparations. The results strongly suggest that a correlation exists between the ability of histamine and its analogs to stimulate isolated rat parietal cell function and their ability to stimulate adenylyl cyclase activity. Potentiation of aminopyrine accumulation in the presence of histamine and carbamylcholine is due to specific receptor effects of each secretagogue.

Cytochalasin inhibition of isolated rat gastric parietal cell function.

Submicrogram concentrations (0.04-0.29 microM) of the microfilament disrupting agents cytochalasins D, E, and B (CD, CE, CB) were shown to inhibit secretagogue-stimulated 14C-aminopyrine accumulation (AP) in isolated rat gastric mucosal parietal cells. The microtubule disrupting agent colchicine had little influence on AP accumulation. Histamine- and dibutyryl cyclic AMP (DbcAMP)-stimulated AP accumulation was inhibited with an order of potency CD greater than CE approximately equal to CB. CB inhibition of these secretagogue actions was, however, only approximately 65-70% of the maximal stimulated response, whereas CD and CE caused 100% inhibition. On the other hand, carbamylcholine-stimulated AP accumulation was inhibited 100% by all cytochalasins tested with an order of potency CD approximately equal to CE greater than CB. These data are discussed in relation to acid secretagogue-induced morphological changes involving actin filament organization in parietal cells.

Histamine regulation of adenylyl cyclase of enriched rat gastric parietal cells.

Rat gastric parietal cells were isolated to 90% or greater purity by discontinuous gradient ultracentrifugation in sucrose-Ficoll. Adenylyl cyclase activity was initiated by histamine in the presence of 5'-guanylyl imidodiphosphate, but not by pentagastrin or acetylcholine. The concentration of histamine needed for half-maximal activation of adenylyl cyclase was 38 microM. The histamine H2-receptor antagonist, metiamide, inhibited histamine (100 microM) activation with a Ki of 0.9 microM. The effects of other histamine agonists and antagonists suggest that presence of H2-type histaminic receptors on parietal cells. Adenylyl cyclase activity was also stimulated by isoproterenol, norepinephrine, and epinephrine, but not by methoxamine. Half-maximal activation by isoproterenol occurred at 0.5 microM. Propranolol, but not phentolamine, prevented activation by these agents, indicating the presence of beta-adrenergic receptors on parietal cells. Prostaglandins E2 stimulated adenylyl cyclase of partially purified (30%), but not the enriched (90%), parietal cell populations.

Isolated parietal cells: [3H]QNB binding to putative cholinergic receptors.

The tritiated muscarinic cholinergic antagonist quinuclidinyl benzilate, [3H]QNB, was used as a direct probe for the detection and characterization of muscarinic cholinergic receptors associated with the particulate fraction of isolated and purified rat gastric muscosal parietal cells. Specific binding is saturable (Bmax = 55 fmol/mg protein, KD = 0.78 nM), shows a single population of binding sites, and has appropriate pharmacological specificity. Nanomolar concentrations of muscarinic cholinergic antagonists, such as atropine and scopolamine, inhibit [3H]QNB binding by 50%, whereas micromolar concentrations are needed for agonists, such as acetylcholine, oxotremorine, and carbamylcholine. Binding is also stereoselective as shown by the more than 1,000-fold difference in inhibitory potencies of the stereoisomers of benzetimide. Noncholinergic agents, including pentagastrin, histamine, and the H2-receptor antagonists cimetidine and metiamide, have little or no effect on [3H]QNB binding at concentrations of 100 microM. These data support the existence of specific parietal cell muscarinic cholinergic receptors with which the secretagogue acetylcholine may directly interact to initiate gastric acid secretion.