Liposome delivery of cyclic AMP-dependent protein kinase inhibitor into intact cells: specific blockade of cyclic AMP-mediated adrenocorticotropin release from mouse anterior pituitary tumor cells.

Insertion of a crude preparation of cyclic AMP (cAMP)-dependent protein kinase inhibitor (PKI) into a cloned mouse anterior pituitary cell line (AtT-20/D16-16) blocked cAMP-mediated hormone release. This was accomplished by developing a technique to incorporate PKI into multicellular cultures. The technique involved the encapsulation of the PKI into liposomes coupled to Protein A (a bacterial protein that binds to the Fc portion of antibodies). Application of such liposomes to AtT-20 cells targeted by pre-treatment with an antiserum against neural cell adhesion molecule (a cell surface glycoprotein expressed by these cells) resulted in the attachment of the liposomes onto the cell surface followed by the delivery of the liposome content into the cells. The AtT-20 cells respond to cAMP-promoting agents such as forskolin by secreting the hormone adrenocorticotropin (ACTH). Liposomes containing PKI and coupled to protein A specifically blocked cAMP-mediated ACTH release from cells treated with anti-N-CAM antibodies. In contrast, the ACTH release response to K+ or phorbol esters does not appear to involve cAMP and was not reduced by such manipulations. The specificity of PKI to block hormone release initiated by one but not by other secretagogues directly links cAMP-dependent protein kinase with the ACTH release process but suggests that there are other mechanisms also involved in stimulus-secretion coupling in corticotrophs.

Stress hormones: their interaction and regulation.

Stress stimulates several adaptive hormonal responses. Prominent among these responses are the secretion of catecholamines from the adrenal medulla, corticosteroids from the adrenal cortex, and adrenocorticotropin from the anterior pituitary. A number of complex interactions are involved in the regulation of these hormones. Glucocorticoids regulate catecholamine biosynthesis in the adrenal medulla and catecholamines stimulate adrenocorticotropin release from the anterior pituitary. In addition, other hormones, including corticotropin-releasing factor, vasoactive intestinal peptide, and arginine vasopressin stimulate while the corticosteroids and somatostatin inhibit adrenocorticotropin secretion. Together these agents appear to determine the complex physiologic responses to a variety of stressors.

Beta-adrenergic mechanism of insulin-induced adrenocorticotropin release from the anterior pituitary.

Intraperitoneal administration of insulin to control rats and to rats with pituitary stalk transections or with lesions of the median eminence resulted in increased plasma adrenocorticotropin (ACTH) levels. The insulin-induced stimulation of ACTH release was blocked in both the control and lesioned animals by prior treatment with either the beta-adrenergic antagonist propranolol or the glucocorticoid analog dexamethasone. The direct application of insulin to primary cultures of the anterior pituitary did not evoke ACTH release or affect the maximal ability of corticotropin-releasing factor or epinephrine to stimulate ACTH secretion. The results suggest that insulin stimulates ACTH release by a mechanism in which catecholamines of peripheral origin act directly on the anterior pituitary.

Molecular biology of opioid receptors.

Opium and its derivatives are potent analgesics that also have many other pharmacological effects in the nervous system. These agents and the endogenous opioid peptides exert their effects by interacting with high-affinity receptors. Complementary DNAs encoding the delta, kappa and mu opioid receptors have been isolated and characterized. These receptors, which are members of the superfamily of seven-transmembrane spanning receptors, share a high degree of amino acid sequence similarity with approximately 50% of the residues being identical. The cloned opioid receptors mediate agonist inhibition of cyclic AMP formation and have pharmacological properties similar to the endogenous proteins. The cloning of these receptors will facilitate the development of new clinically useful compounds as well as studies of the molecular basis of tolerance and drug addiction.

Molecular biology of somatostatin receptors.

The neuropeptide somatostatin (SRIF) has diverse physiological actions in the brain and endocrine organs. A family of SRIF receptors has recently been cloned that may mediate the distinct biological effects of SRIF. These receptors have a high degree of amino acid sequence similarity among themselves, but their sequences are different from any other receptors, indicating that they represent a distinct neurotransmitter receptor subfamily. The availability of the cloned receptors will now allow for detailed structure-function analysis of SRIF receptors and will facilitate development of subtype-selective agonists and antagonists that could be useful in the treatment of central nervous system and endocrine disorders.

Opioid and cannabinoid receptors.

Opioids and cannabinoids are two major classes of drugs with important clinical uses as well as significant side effects. Recently, the three major subtypes of opioid receptors, delta, kappa and mu, have been cloned. Both the endogenous cannabinoids and their receptors have also recently been cloned. These advances are facilitating attempts to understand the structural features of these receptors that are involved in their functioning, which should lead to the development of new and improved clinically useful opioids and cannabinoid-like drugs.

Endomorphin-1: induction of motor behavior and lack of receptor desensitization.

The endomorphins are recently discovered endogenous agonists for the mu-opioid receptor (Zadina et al., 1997). Endomorphins produce analgesia; however, their role in other brain functions has not been elucidated. We have investigated the behavioral effects of endomorphin-1 in the globus pallidus, a brain region that is rich in mu-opioid receptors and involved in motor control. Bilateral administration of endomorphin-1 in the globus pallidus of rats induced orofacial dyskinesia. This effect was dose-dependent and at the highest dose tested (18 pmol per side) was sustained during the 60 min of observation, indicating that endomorphin-1 does not induce rapid desensitization of this motor response. In agreement with a lack of desensitization of mu-opioid receptors, 3 hr of continuous exposure of the cloned mu receptor to endomorphin-1 did not diminish the subsequent ability of the agonist to inhibit adenylate cyclase activity in cells expressing the cloned mu-opioid receptor. Confirming the involvement of mu-opioid receptors, the behavioral effect of endomorphin-1 in the globus pallidus was blocked by the opioid antagonist naloxone and the mu-selective peptide antagonist Cys(2)-Tyr(3)-Orn(5)-Pen(7) amide (CTOP). Furthermore, the selective mu receptor agonist [d-Ala(2)-N-Me-Phe(4)-Glycol(5)]-enkephalin (DAMGO) also stimulated orofacial dyskinesia when infused into the globus pallidus, albeit transiently. Our findings suggest that endogenous mu agonists may play a role in hyperkinetic movement disorders by inducing sustained activation of pallidal opioid receptors.

Structure-function analysis of the cloned opiate receptors: peptide and small molecule interactions.

Opiate receptors mediate the physiological actions of opioid peptides and the clinical effects of the synthetic opioid agonists and antagonists. Site-directed mutagenesis studies have revealed regions of opiate receptors that are essential for ligand recognition, and this could aid the design of more selective opioid ligands.

Somatostatin receptor activation of cellular effector systems.

Somatostatin (SRIF) induces its multiple biological actions by interacting with a family of receptors, referred to as SSTR1-SSTR5. These receptors are capable of associating with particular guanine nucleotide binding proteins to couple the receptors to distinct cellular effector systems. Therefore, G proteins have an important role in directing SRIF signalling and may provide the molecular basis for the diverse cellular actions of SRIF.

Somatostatin receptors, an expanding gene family: cloning and functional characterization of human SSTR3, a protein coupled to adenylyl cyclase.

We previously reported the cloning of two distinct somatostatin receptor (SSTR) subtypes, SSTR1 and SSTR2. Although both SSTR1 and SSTR2 bound somatostatin specifically and with high affinity, neither was coupled to adenylyl cyclase, a major cellular effector of somatostatin's actions. Here we report the cloning and functional characterization of a third member of the SSTR family. Human SSTR3 is a protein of 418 amino acids and has 45% and 46% identity with human SSTR1 and SSTR2, respectively. RNA blotting studies showed that SSTR3 mRNA could be readily detected in brain and pancreatic islets. The pharmacological properties of human SSTR3 were characterized by transiently expressing the human SSTR3 gene in COS-1 cells. Membranes from cells expressing human SSTR3 bound the somatostatin agonist [125I]CGP 23996 specifically and with high affinity, with a rank order of potency of somatostatin-28 = CGP 23996 > somatostatin-14 > SMS-201-995. Studies using cells transiently coexpressing the human dopamine D1 receptor and human SSTR3 showed that somatostatin was able to inhibit dopamine-stimulated cAMP formation in a dose-dependent manner, indicating that SSTR3 was functionally coupled to adenylyl cyclase. These results indicate that the diverse biological effects of somatostatin are mediated by a family of receptor with distinct, but overlapping, tissue distributions, unique pharmacological properties, and potentially different functions.

Multiple mechanisms of somatostatin inhibition of adrenocorticotropin release from mouse anterior pituitary tumor cells.

The release of ACTH from a clonal cell line of the mouse anterior pituitary (AtT-20/D16-16) can be stimulated by forskolin, 8-bromo-cAMP, and K+. SRIF and its structurally related analogs are very potent inhibitors of the ACTH release response to these secretagogues. The potency of SRIF, its analogs, and somatostatin-28 to inhibit stimulated ACTH release is relatively the same for each of these three secretagogues. The mechanisms by which SRIF regulates the secretion of ACTH can be differentiated by various pharmacological manipulations. Pretreatment of AtT-20 cells with SRIF (10(-7) M) desensitizes SRIF's inhibition of forskolin but not K+ or 8-bromo-cAMP-stimulated ACTH release. Pertussis toxin pretreatment abolishes SRIF's inhibition of forskolin-stimulated ACTH release but not SRIF's inhibition of the ACTH release response to K+ or 8-bromo-cAMP. In contrast, increasing the calcium concentration in the medium reduces SRIF's inhibition of K+ but not forskolin or 8-bromo-cAMP-stimulated ACTH release. These results suggest that SRIF regulates ACTH release from AtT-20 cells through multiple mechanisms. If SRIF acts through a single receptor to produce its effects on ACTH release, then the data imply that the SRIF receptor is coupled to more than one second messenger system.

Prolonged somatostatin pretreatment desensitizes somatostatin's inhibition of receptor-mediated release of adrenocorticotropin hormone and sensitizes adenylate cyclase.

Addition of somatostatin-14 (SRIF) inhibits corticotropin releasing factor (CRF) and forskolin-stimulated cyclic AMP formation and ACTH release from tumor cells of the mouse anterior pituitary (AtT-20/D16-16). After long-term pretreatment of these cells with SRIF, the ability of SRIF to inhibit CRF and forskolin-stimulated cyclic AMP accumulation or ACTH secretion is markedly reduced. SRIF pretreatment also increases the formation of cyclic AMP in response to forskolin. This increase is delayed in onset, slow to recover, and blocked by the protein synthesis inhibitor, cycloheximide. SRIF pretreatment did not affect basal cyclic AMP and cyclic GMP levels or phosphodiesterase activity. It is proposed that prolonged treatment of AtT-20 cells with SRIF desensitizes SRIF receptors and induces a compensatory sensitization of adenylate cyclase through a process requiring protein synthesis.

Adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase activity in rodent pituitary tissue: possible role in cAMP-dependent hormone secretion.

A cAMP-dependent protein kinase occurs in the intermediate lobe of the rat pituitary gland and the ACTH-secreting tumor AtT-20/D16-16 derived from the mouse pituitary gland. Exposure of either tissue to drugs increasing cAMP production and hormone release (forskolin, cholera toxin, or isoproterenol in the case of the intermediate lobe; forskolin or isoproterenol in the case of the AtT-20 cells) increases the cAMP-dependent protein kinase activity of a tissue homogenate in the absence, but not in the presence, of added cAMP. The potencies of these drugs to induce changes in the protein kinase activity ratio (i.e. enzyme activity in the absence of cAMP to enzyme activity in the presence of 3 microM cAMP) are comparable with their potencies as stimulants of hormone secretion. In either tissue, A23187, a calcium ionophore that stimulates hormone release but not cAMP production, does not change the protein kinase activity ratio. In the case of the AtT-20 cells, dexamethasone blocks the release of ACTH simulated by either isoproterenol or forskolin, but does not alter the enhancement of protein kinase activity induced by these drugs. Conversely, dexamethasone does not block the A23187-stimulated release of ACTH. The data suggest that cAMP modulates (but does not trigger) hormone secretion from the rodent pituitary gland by a mechanism involving activation of the cAMP-dependent protein kinase. Several possible sites for this modulatory effect of cAMP are discussed.

Forskolin enhances basal and potassium-evoked hormone release from normal and malignant pituitary tissue: the role of calcium.

The release of immunoreactive ACTH (IR-ACTH) from AtT-20 pituitary tumor cells was transiently increased by exposure to an elevated concentration of potassium ion in an osmotically balanced extracellular medium. With the calcium-sensitive dye Quin 2, the concentration of free cytosolic calcium (Cai) in the AtT-20 tumor was determined to be 115 nM. Challenge of these cells with 60 mM potassium in an osmotically balanced salt solution raised the concentration of Cai to 246 nM. This is in accord with the view that agents promoting calcium entry into pituitary cells trigger hormone secretion. Addition of forskolin to the extracellular medium caused a sustained release of IR-ACTH from AtT-20 tumor cells. Challenge with forskolin (10 microM) increased the concentration of Cai to 149 nM. This observation is also in accord with the view that calcium entry is a necessary and sufficient stimulus to trigger hormone secretion from the anterior pituitary lobe. Exposure of cells to forskolin (10 microM) before a potassium challenge increased the quantity of IR-ACTH released in response to potassium, but did not alter the minute by minute time course of the response to this ion. Forskolin pretreatment did not alter the potassium-evoked rise in Cai concentration. This observation suggests that the magnitude of the secretory response of the pituitary gland can be enhanced by agents other than those promoting an increase in Cai. After exposure of the tumor cells to potassium for a sufficient time to permit the rate of release of hormone to return to the basal value, forskolin could still stimulate the release of hormone from the tumor cells. Under these circumstances, forskolin did not increase the concentration of Cai. This observation suggests that pituitary hormone secretion can be initiated by a factor(s) other than an acute change in the Cai concentration. Both forskolin and 8-bromo-cAMP stimulated hormone secretion from dispersed melanotrophs and potentiated the potassium-evoked secretory response of these cells. Neither compound affected the apparent time course of the response to potassium. These observations suggest that the effects of forskolin and potassium on the AtT-20 tumor cell may use mechanisms occurring in normal pituitary cells.

Differential internalization of somatostatin in COS-7 cells transfected with SST1 and SST2 receptor subtypes: a confocal microscopic study using novel fluorescent somatostatin derivatives.

A growing body of evidence suggests that neuropeptide binding to G protein-linked receptors may result in internalization of receptor-ligand complexes, followed by intracellular mobilization and degradation of the ligand into its target cells. Because of discrepant results in the literature concerning the occurrence of such a mechanism for the tetradecapeptide somatostatin (SRIF), we have reinvestigated this question by comparing the binding and internalization of iodinated and fluorescent derivatives of the metabolically stable analog of SRIF, [D-Trp8]SRIF, in COS-7 cells transfected with complementary DNA encoding the sst1 or sst2A receptor subtype. A series of fluoresceinyl and Bodipy fluorescent derivatives of [D-Trp8]SRIF-14 was purified by HPLC, analyzed for purity by mass spectrometry, and tested for biological activity in a membrane binding assay. Of the six compounds tested, fluoresceinyl and Bodipy derivatives labeled in position alpha (fluo-SRIF) retained high affinity for SRIF receptors. COS-7 cells transfected with complementary DNA encoding either sst1 or sst2A receptors both displayed specific, high affinity binding of iodinated and fluo-SRIF. At 4 C, the labeling was confined to the cell surface in both cell types, as indicated by the fact that it was entirely removable by a hypertonic acid wash and assumed a pericellular distribution in the confocal microscope. At 37 C, the fate of specifically bound ligand varied markedly according to the type of receptor transfected. In cells encoding the sst1 receptor, approximately 20% of specifically bound ligand was recovered in the acid-resistant (i.e. intracellular) fraction. This fraction remained clustered at the periphery of the cell, suggesting that it was being sequestered either within or immediately beneath the plasma membrane. By contrast, in cells transfected with sst2A receptors, up to 75% of specifically bound ligand was recovered inside the cells, where it clustered into small endosome-like particles. These particles increased in size and moved toward the nucleus with time, suggestive of receptor-ligand complexes proceeding down the endocytic pathway. These results demonstrate that neuropeptides may be processed differently depending on the subtype of receptor expressed in their target cells and suggest that these different processing patterns may reflect different modes of sensitization/desensitization and recycling of the receptors, and thereby of transmembrane signaling.

Expression of mu opioid receptor mRNA in rat brain: an in situ hybridization study at the single cell level.

The mu (mu) opioid receptors, which mediate the effects of morphine, are widely distributed in brain. We have examined the distribution of mRNA encoding a mu opioid receptor in rat brain with in situ hybridization histochemistry at the single-cell level to obtain information about the cell types synthesizing this receptor. Only neurons, not glia, were labeled in discrete brain regions. High levels of labeling were detected in the thalamus, striosomes of the caudate-putamen, globus pallidus, and brain regions involved in nociception, arousal, respiratory control, and, possibly, addiction. The general distribution of the receptor mRNA paralleled that of mu opioid binding sites with some notable exceptions. These include the cerebral cortex, which contains binding sites, but very few labeled neurons. No labeling was observed in the cerebellum, a region devoid of mu binding sites. Three main findings emerged from these experiments: 1) the mRNA was present in regions mediating both the therapeutic (analgesia) and the unwanted (respiratory depression, addiction) effects of morphine, 2) the mRNA was very densely expressed by neurons known to receive dense enkephalin-containing inputs, and 3) the dissociation between the presence of binding sites and absence of mRNA in some brain regions supports a presynaptic localization of mu opioid receptors in these areas. Alternatively, other subtypes of mu opioid receptors may be encoded by a different mRNA. These results provide new insights into the receptor types and neuronal circuits involved in the effects of endogenous opioids and morphine.

Lanthionine-somatostatin analogs: synthesis, characterization, biological activity, and enzymatic stability studies.

A series of cyclic somatostatin analogs containing a lanthionine bridge have been subjected to studies of structure-activity relationships. A direct synthesis of the thioether bridged analog (1) of sandostatin (SMS 201,995) and several lanthionine hexa-, hepta-, and octapeptides was carried out by using the method of cyclization on an oxime resin (PCOR) followed by condensation reactions in solution. The structures of the target peptides were analyzed by liquid secondary ion mass spectrometry (LSIMS) and subjected to high-energy collision-induced dissociation (CID) studies after opening of the peptide ring by proteolytic cleavage. The biological activities of these compounds have been evaluated by assaying their inhibitory potencies for the release of growth hormone (GH) from primary cultures of rat anterior pituitary cells, as well as by their binding affinities to cloned somatostatin receptors (SSTR1-5). The structural modification of sandostatin by introducing a lanthionine bridge resulted in a significantly increased receptor binding selectivity. The lanthionine octapeptide with C-terminal Thr-ol (1) showed similar high affinity for rat SSTR5 compared to somatostatin[1-14] and sandostatin. However, it exhibits about 50 times weaker binding affinity for mSSTR2b than sandostatin. Similarly, the lanthionine octapeptide with the C-terminal Thr-NH2 residue (2) has higher affinity for rSSTR5 than for mSSTR2B. Both peptides (compounds 1 and 2) have much lower potencies for inhibition of growth hormone secretion than sandostatin. This is consistent with their affinities to SSTR2, the receptor which is believed to be linked to the inhibition of growth hormone release by somatostatin and its analogs. The metabolic stability of lanthionine-sandostatin and sandostatin have been studied in rat brain homogenates. Although both compounds have a high stability toward enzymatic degradation, the lanthionine analog has a 2.4 times longer half-life than sandostatin. The main metabolites of both compounds have been isolated and identified by using an in vivo technique (cerebral microdialysis) and mass spectrometry.

Molecular properties of somatostatin receptors.

The neuropeptide somatostatin is widely distributed in the central nervous system and in peripheral tissues and may be involved in the regulation of a number of physiological functions including movement and cognition. Somatostatin may also have a role in the development of the central nervous system, in particular, the cerebellum and spinal cord. Somatostatin induces its actions by interacting with a family of membrane associated receptors. Recently, five somatostatin receptors have been cloned and referred to as SSTR1-SSTR5. The distribution of the expression of the mRNAs for these receptors are distinct but overlapping. Preliminary pharmacological analysis of these receptors may lead to the development of selective ligands at these receptors. These compounds may be useful in identifying the selective functions of these receptor subtypes. Some somatostatin analogues have antiproliferative actions and are used presently to treat carcinoids. Development of subtype selective somatostatin analogues could be helpful in further identifying somatostatin receptor-expressing tumors and in the treatment of cancer. The cloning of these receptors has now opened up the possibility of more clearly investigating the functions of somatostatin in the brain and peripheral tissues and will facilitate the generation of new somatostatin drugs that may be employed for the treatment of a number of diseases.

Differential regulation of the cloned kappa and mu opioid receptors.

To directly compare the regulation of the cloned kappa and mu opioid receptor, we expressed them in the same cells, the mouse anterior pituitary cell line AtT-20. The coupling of an endogenous somatostatin receptor to adenylyl cyclase and an inward rectifier K+ current has been well characterized in these cells, enabling us to do parallel studies comparing the regulation of both the kappa and the mu receptor to this somatostatin receptor. We show that the kappa receptor readily uncoupled from the K+ current and from adenylyl cyclase after a 1 h pretreatment with agonist, as indicated by the loss in the ability of the agonist to induce a functional response. The desensitization of the kappa receptor was homologous, as the ability of somatostatin to mediate inhibition of adenylyl cyclase or potentiation of the K+ current was not altered by kappa receptor desensitization. The mu receptor uncoupled from the K+ current but not adenylyl cyclase after a 1 h pretreatment with agonist. Somatostatin was no longer able to potentiate the K+ current after mu receptor desensitization, thus this desensitization was heterologous. Interestingly, pretreatment with a somatostatin agonist caused uncoupling of the mu receptor but not the kappa receptor from the K+ current. These results show that in the same cell line, after a 1 h pretreatment with agonist, the kappa receptor displays homologous regulation, whereas the mu receptor undergoes only a heterologous form of desensitization. mu receptor desensitization may lead to the alterations of diverse downstream events, whereas kappa receptor regulation apparently occurs at the level of the receptor itself. Broad alterations of non-opioid systems by the mu receptor could be relevant to the addictive properties of mu agonists. Comparison of kappa and mu receptor regulation may help define the properties of the mu receptor which are important in the development of addiction, tolerance, and withdrawal to opioid drugs. These are the first studies to directly compare the coupling of the kappa and mu receptors to two different effectors in the same mammalian expression system.

Evidence that a novel somatostatin receptor couples to an inward rectifier potassium current in AtT-20 cells.

The recent cloning of five somatostatin receptors has made it possible to begin screening for selective ligands in order to begin characterization of these receptor subtypes expressed endogenously. We have recently reported the characterization of ligands selective for SSTR2 and SSTR5 [Raynor K. et al. (1993) Molec. Pharmac. 43, 838-844; 44, 385-392]. Both of these somatostatin receptor subtypes are endogenously expressed in the mouse pituitary cell line AtT-20 [O'Carroll A.-M. et al. (1992) Molec. Pharmac. 42, 939-946; Patel Y. C. et al. (1994) J. biol. Chem. 269, 1506-1509; Tallent M. et al. (1996) Neuroscience 71, 1073-1081]. Using these selective ligands, as well as other somatostatin analogs, we have characterized the somatostatin receptor which couples to the inward rectifier K+ current in AtT-20 cells. This receptor is sensitive to hexapeptide analogs of somatostatin, but insensitive to octapeptide analogs. This pharmacological profile is distinct from any of the cloned somatostatin receptors and therefore may represent a novel receptor. Somatostatin has been shown to potentiate an inward rectifying K+ channel in many different types of neuronal and non-neuronal cells. The activation of this current is thought to be an important mechanism by which somatostatin inhibits neuronal firing and decreases neurotransmitter and hormone release [Mihara S. et al. (1987) J. Physiol. 390, 335-355]. Therefore, the novel somatostatin receptor coupling to the inward rectifier in AtT-20 cells may be important in somatostatin's role in regulating neurotransmission and hormone release.