Biosynthetic response of mouse intermediate pituitary gland to induced drinking and dehydration

There are indications that the intermediate lobe peptide alpha-MSH is involved in the regulation of the hydromineral balance in mice and other mammals. The purpose of our studies was to determine whether manipulation of this balance in the mouse could lead to changes in either the rate of POMC biosynthesis in the pars intermedia or to changes in the direction of the processing of the precursor protein to form bioactive peptides. The results show that excess drinking, induced by substitution of drinking water by a 5% glucose solution, causes a rapid increase in POMC synthesis, whereas dehydration has the opposite effect; no evidence could be found that the above treatments have any effect on the processing of POMC, although strain differences were found in level of N-terminal acetylation of newly synthesized melanotropins and endorphins. The changes in various parameters of the hydromineral balance of the animals are consistent with the concept that peptides of the pars intermedia may be involved in regulating plasma aldosterone levels under severe conditions of low plasma sodium concentration.     

Intermediate lobe of the amphibian pituitary gland: an endocrine gland with multiple secretions and under multi-hormonal control

The cells of the frog pars intermedia synthesize a 36 000 (36K) protein called proopiomelanocortin (POMC). After [3H]glucosamine incorporation, separation of newly synthesized products by SDS-polyacrylamide gel electrophoresis showed that this 36K protein was glycosylated. Tryptic mapping revealed only one site of glycosylation and showed that the carbohydrate side-chain was located in the N-terminal region of POMC. The 36K protein was not released by the melanotrophs, but it generated, through specific intracellular proteolytic cleavage, a number of smaller peptides which were subsequently released. These peptides were identified by various methods including selective amino-acid incorporation, HPLC purification, acid-urea gel electrophoresis, tryptic and chymotryptic mapping, assay of melanotropic activity, radioimmunoassays and immunoprecipitations. Some of the newly synthesized N-terminal (18K) fragment of the POMC was secreted intact while a portion of it was further processed, via an intermediate peptide, to give mature gamma-MSH. All three of these peptides were glycosylated. In addition, the mature peptide (gamma-MSH) exhibited a low but significant melanotropic activity. The C-terminal portion of the prohormone was very rapidly processed to give des N alpha-acetyl alpha-MSH, corticotropin-like-intermediate lobe peptide (CLIP) and beta-endorphin. Authentic alpha-MSH was always absent in cellular extracts: acetylation to give rise to alpha-MSH was a late enzymatic process strictly linked to hormonal release. Since acetylation of alpha-MSH is required for full biological activity of this peptide, it is possible to conceive that this later step could be under neuroendocrine control. Using the perifusion technique we have been able to show the complexity of the control mechanisms regulating amphibian melanotrophs. It is generally accepted that the aminergic innervation of the intermediate lobe of the pituitary is involved in the hypothalamic control of melanotropin release. We have demonstrated that, in amphibians, dopamine inhibits alpha-MSH secretion through D2-type dopaminergic receptors whereas norepinephrine and (or) epinephrine stimulate alpha-MSH secretion via beta-adrenergic receptors. The existence of peptidergic fibers within parenchymal cells of the pars intermedia has been demonstrated. Evidence for TRH-containing fibers has been obtained by immunohistochemistry. Using a specific radioimmunoassay for TRH, we have confirmed the presence of TRH in the neurointermediate lobe of the frog. We have shown that TRH is a powerful MSH-releasing factor in these animals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and glycosylation of proopiomelanocortin by a Cushing tumor

ACTH derives from a larger protein, proopiomelanocortin (POMC), which is also the precursor to lipotropin, endorphin, melanotropin, and other peptides. The sequence of steps by which POMC is processed to ACTH has been delineated in detail in cultured mouse tumor cells, but little is known about these steps in human pituitary adenomas. Synthesis and glycosylation of POMC were studied in a pituitary adenoma causing Cushing disease and in adjacent tissue by incubating intact tissue explants in medium containing [35S]methionine or [3H]glucosamine. Labeled tissue was analyzed by two-dimensional gel electrophoresis, and a spot cut from these gels was analyzed by paper electrophoresis of tryptic digests. The location of the spot and the patterns of its [35S]methionine-labeled tryptic fragments resembled those found previously in other ACTH-secreting tissues and hence this spot appears to be POMC. Both the tumor and the adjacent pituitary produced this POMC, although tumor production of POMC was greater. The electrophoretic patterns of the putative POMC tryptic peptides labeled with [35S]methionine from this tumor are similar to the patterns of POMC tryptic peptides from a Nelson tumor and an ectopic ACTH- producing tumor when analyzed by the same techniques. This suggests that the excess ACTH in all three diseases arises from the same precursor POMC molecule. Two-dimensional gel electrophoresis of [3H]glucosamine-labeled tumor suggests that the first proteolytic cleavage occurs between the alpha ACTH-(1-39) and beta-lipotropin sequences and that a subsequent cleavage occurs between the N-terminal region of POMC and ACTH. Paper electrophoresis of [3H]glucosamine-labeled tryptic glycopeptides reveals at least three radioactive peaks, suggesting that the two known glycosylation sites in human POMC may be variably glycosylated.     

Plasma immunoreactive proopiomelanocortin peptides and cortisol in normal dogs and dogs with Cushing's syndrome: diurnal rhythm and responses to various stimuli

We have studied the diurnal rhythm of pars distalis and pars intermedia-type immunoreactive (IR)-POMC peptides and cortisol in 3 normal dogs and 1 dog with Cushing's syndrome and have documented the responses to a variety of agents in 42 dogs with Cushing's disease, 2 of which were known or presumed to have pars intermedia tumors and another of which had both pars distalis and pars intermedia adenomas, and in 20 dogs with adrenocortical adenomas causing Cushing's syndrome. The normal dogs did not have a diurnal plasma POMC peptide rhythm; the dog with Cushing's disease appeared to have a similar number of secretory episodes of increased amplitude. Plasma POMC peptides and cortisol in animals with Cushing's disease did not suppress normally with low dose dexamethasone. Five animals with Cushing's disease did suppress with high dose dexamethasone, the dog with dual adenomas suppressed only partially, and 1 dog with a pars intermedia adenoma did not suppress at all. The response to insulin-induced hypoglycemia was similar in normal dogs and 4 dogs with Cushing's disease, but 3 animals with adrenal tumors did not respond. The response to metyrapone was normal in 6 dogs with Cushing's disease and, surprisingly, in 1 with adrenal tumor. Arginine vasopressin stimulated POMC peptide secretion in normal and 6 Cushing's dogs, as well as alpha MSH, a pars intermedia-type POMC peptide, in a dog presumed to have a pars intermedia tumor. Ovine CRF stimulated pars distalis-type POMC peptide secretion in normal dogs and 17 dogs with Cushing's disease, but not in 15 dogs with adrenal tumor; IR-alpha MSH was unaffected. TRH appeared to stimulate IR-ACTH in normal animals, but not in those with Cushing's disease. Dopamine had no apparent effect in 2 normal and 1 Cushing's dogs. Initial plasma disappearance t1/2 values of IR-ACTH and lipotropin were 22-27 min. In summary, responses in normal and Cushing's dogs were generally what would be predicted from previous human and animal studies, but some of those in animals with pars intermedia tumors and even in normal dogs were different from what had been anticipated. Canine Cushing's syndrome provides an interesting model for an uncommon human disorder.     

The development of the pars intermedia and its role in the regulation of dermal melanophores in the larvae of the amphibian Xenopus laevis

The ontogenesis of biosynthesis of pro-opiomelanocortin (POMC)-related peptides in the pars intermedia of Xenopus laevis tadpoles was studied. The results were related to the capacity of the animal to adapt to background color through regulation of pigment dispersion in dermal melanophores. Using immunocytochemical techniques with antisera to alpha-melanophore-stimulating hormone (alpha-MSH), it was revealed that this peptide first appeared at developmental stage 37/38, just prior to the animal's ability to adapt to background. It was shown that pigment dispersion in melanophores between stages 33 and 39 was not dependent on melanotropins of pituitary origin. Using in vitro biosynthetic studies it was possible to follow POMC biosynthetic activity, its processing and the release of peptides from stage 48 onward. Among the newly synthesized peptides observed were a gamma 3-MSH-like peptide, des-N-alpha-acetyl-alpha-MSH, alpha-MSH, and two endorphin-like peptides. By stage 57 a biosynthetic pattern almost identical to that of the adult pars intermedia had evolved. It was concluded that stage 39/40 is a critical stage in the simultaneous development of a number of the components involved in the neuroendocrine control of background adaptation.     

Effects of labor on pituitary expression of proopiomelanocortin, prohormone convertase (PC)-1, PC-2, and glucocorticoid receptor mRNA in fetal sheep

We hypothesized that the concurrent prepartum rise in adrenocorticotropic hormone (ACTH) and cortisol in the plasma of fetal sheep might be attributable to altered expression of pituitary endoproteases, prohormone convertase (PC)-1, and PC-2, or to changes in pituitary expression of glucocorticoid receptor (GR) that would influence negative feedback potential. We obtained pituitary tissue from fetal sheep during late pregnancy (d 100–d 145, term) and at precise times during the process of labor and used in situ hybridization to localize and quantify mRNA levels. Proopiomelanocortin (POMC) mRNA was regionally distributed (pars intermedia > inferior pars distalis > superior pars distalis) and increased within the pars distalis during late pregnancy and with labor. At term, levels of PC-1 and PC-2 mRNA were higher in the pars intermedia than pars distalis; PC-1 but not PC-2 in the pars distalis increased with gestational age, although it did not change further at labor. GR mRNA levels in the pars distalis increased between d 135 and term, then decreased during labor. We suggest that the concomitant rise in plasma ACTH and cortisol of fetal sheep during late gestation may be attributable, in part, to increased expression of PC-1 leading to increased POMC processing. Furthermore, the negative feedback effects of cortisol on pituitary POMC synthesis and/or ACTH release during active parturition may be lessened by downregulation of anterior pituitary GR.     

Distribution of immunoreactive sites for several components of pro-opiocortin in the pituitary and brain of adult lampreys, Petromyzon marinus and Entosphenus tridentatus

The occurrence and localization of molecular components of pro-opiocortin in the pituitary and brain of two species of adult lamprey, Petromyzon marinus and Entosphenus tridentatus, were studied immunocytochemically using antisera generated against human pro-gamma-MSH (N-terminal fragment 1Trp to 71Gly of pro-opiocortin), porcine ACTH, alpha-MSH, human beta-endorphin, gamma-endorphin, and methionine enkephalin. (1) In both species of lamprey most cells of the rostral pars distalis and some cells of the caudal pars distalis contained Met-enkephalin-like immunoreactivity. Some of these cells also contained gamma-endorphin-like immunoreactivity. After preabsorbing the antisera with their corresponding antigens or related peptides, the Met-enkephalin/gamma-endorphin-like material was found to be related to Met-enkephalin, but not identical with either Met-enkephalin or gamma-endorphin. However, results of anti-pro-gamma-MSH, anti-ACTH, anti-alpha-MSH, or anti-beta-endorphin were consistently negative in the pars distalis of both lamprey species. (2) Immunoreaction to anti-Met-enkephalin was found in some cells of the pars intermedia in both species of lamprey. Although the positive reaction had been eliminated by preabsorption with synthetic Met-enkephalin, the diffuseness of the positive stain in the pars intermedia cells resembled an artifactual cross-reaction rather than a specific reaction. In P. marinus, but not in E. tridentatus, similar inconsistent and questionable immunoreactions corresponding to ACTH and alpha-MSH also occurred in some pars intermedia cells. Results of other antisera (anti-pro-gamma-MSH, anti-beta-endorphin, or anti-gamma-endorphin) were consistently negative in the pars intermedia of both lamprey species. (3) In both species of lamprey beta-endorphin-like material was found in the hypothalamus. In E. tridentatus only Met-enkephalin-like material was observed in the hypothalamus, and these two substances were distributed in different neuronal elements. After application of anti-pro-gamma-MSH, anti-ACTH, anti-alpha-MSH, or anti-gamma-endorphin, no positive reaction was found in the brain of either species of lamprey. These findings suggest that if a pro-opiocortin-related prohormone exists in the lamprey, it may be chemically different from those of more advanced vertebrates, and it clearly differs in distribution between the brain and parts of the pituitary gland.     

Plasma immunoreactive proopiomelanocortin peptides and cortisol in normal dogs and dogs with Addison's disease and Cushing's syndrome: basal concentrations

We measured basal plasma concentrations of the immunoreactive (IR) proopiomelanocortin (POMC)-derived peptides ACTH, beta-lipotropin (beta LPH), beta-endorphin (beta END), and alpha MSH in 160 normal dogs, 32 dogs with Addison's disease, 42 dogs with adrenocortical tumors causing Cushing's syndrome, and 169 dogs with pituitary-dependent Cushing's disease. In normal dogs, plasma IR-POMC peptide levels were similar to those in man, except that IR-alpha MSH, a pars intermedia POMC product, was readily detected. In Addisonian dogs, plasma cortisol was decreased, and the IR-POMC peptides were increased, except for IR-alpha MSH, which was normal. In 7 Addisonian dogs given dexamethasone, elevated plasma IR-ACTH, beta LPH, and beta END levels fell dramatically. In dogs with Cushing's syndrome due to adrenal tumors, plasma IR-ACTH, beta LPH, and beta END were decreased, and cortisol was increased, but IR-alpha MSH was normal. Dogs with Cushing's disease due to pars distalis tumors had elevated plasma IR-ACTH, beta LPH, beta END, and cortisol, but normal IR-alpha MSH; their plasma cortisol was suppressed by dexamethasone. There appeared to be 2 types of pars intermedia tumors causing Cushing's disease: 1 dexamethasone nonsuppressible and with disproportionately high plasma IR-alpha MSH levels, the other relatively dexamethasone suppressible and with normal to slightly elevated IR-alpha MSH levels. These 2 pars intermedia tumor types may arise from 2 distinct normal canine pars intermedia cell types. Canine Cushing's disease may provide a useful model for variants of the disorder in man.     

Genetic modifications of mouse proopiomelanocortin peptide processing

Pro-opiomelanocortin (POMC) is a prohormone which undergoes extensive tissue and cell specific post-translational processing producing a number of active peptides with diverse biological roles ranging from control of adrenal function to pigmentation to the regulation of feeding. One approach to unraveling the complexities of the POMC system is to engineer mouse mutants which lack specific POMC peptides. We describe here the design, generation, validation, and preliminary analysis of one such partial POMC mutant specifically lacking α-MSH. In contrast to POMC null mutant mice, mice lacking α-MSH in the presence of all other POMC peptides maintain adrenal structures and produce corticosterone comparable to wildtype littermates; however, they still have decreased levels of aldosterone, as found in POMC null mutant mice. Our findings demonstrate that α-MSH is not needed for maintenance of adrenal structure or for corticosterone production, but is needed for aldosterone production. These data demonstrate that mouse strains generated with precise genetic modifications of POMC peptide processing can answer questions about POMC peptide function. Further analysis of this and additional strains of mice with modified POMC peptide processing patterns will open up a novel avenue for studying the roles of individual POMC peptides.     

Identification of beta-endorphins in the pituitary gland and blood plasma of the common carp (Cyprinus carpio)

Carp beta-endorphin is posttranslationally modified by N-terminal acetylation and C-terminal cleavage. These processes determine the biological activity of the beta-endorphins. Forms of beta-endorphin were identified in the pars intermedia and the pars distalis of the pituitary gland of the common carp (Cyprinus carpio), as well as the forms released in vitro and into the blood. After separation and quantitation by high performance liquid chromatography (HPLC) coupled with radioimmunoassay, the beta-endorphin immunoreactive products were identified by electrospray ionisation mass spectrometry and peptide sequencing. The release of beta-endorphins by the pituitary gland was studied after stimulation with corticotrophin-releasing factor (CRF) in vitro. In the pars intermedia, eight N-acetylated truncated forms were identified. Full length N-acetyl beta-endorphin(1-33) coeluted with N-acetyl beta-endorphin(1-29) and these forms together amounted to over 50% of total immunoreactivity. These products were partially processed to N-acetyl betaendorphin(1-15) (30.8% of total immunoreactivity) and N-acetyl beta-endorphin(1-10) (3.1%) via two different cleavage pathways. The acetylated carp homologues of mammalian alpha- and gamma-endorphin were also found. N-acetyl beta-endorphin(1-15) and (1-29) and/or (1-33) were the major products to be released in vitro, and were the only acetylated beta-endorphins found in blood plasma, although never together. CRF stimulated the release of opioid beta-endorphin from the pars distalis. This non-acetylated beta-endorphin represents the full length peptide and is the most abundant form in plasma.     

In vivo biosynthesis of melanotropins and related peptides in the pars intermedia of Xenopus laevis

To study in vivo biosynthesis of pars intermedia peptides in Xenopus laevis, [3H]lysine was administered by an osmotic minipump via a cannula inserted near the pituitary gland. Following extraction of the neurointermediate lobe, high-performance liquid chromatography was used to separate the newly synthesized peptides. In black-background adapted animals, [3H]lysine was incorporated into a number of peptides. The elution characteristics of these peptides corresponded exactly with those of peptides synthesized during in vitro incubation of neurointermediate lobes, and which were identified as des-N alpha-acetyl-alpha-MSH, a gamma-MSH-like peptide, two corticotropin-like intermediate lobe peptides, and two forms of endorphin. In white-background adapted Xenopus, practically no synthesis of pars intermedia peptides occurred. Transfer of black-adapted toads to a white background at the beginning of infusion led to storage of newly synthesized peptides. When such animals were maintained on a white background for 10 days, des-N alpha-acetyl-alpha-MSH, but not alpha-MSH, was present in the pars intermedia; this supports the notion that des-N alpha-acetyl-alpha-MSH constitutes the "storage form" of alpha-MSH.     

PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues.

A recombinant vaccinia virus vector was used to coexpress the two candidate mouse prohormone convertases, PC1 and PC2, together with mouse proopiomelanocortin (POMC) in the constitutively secreting cell line BSC-40 and in the endocrine tissue-derived cell lines PC12 and AtT-20, which exhibit regulated secretion. Monitoring of POMC processing demonstrated the distinct cleavage specificities of PC1 and PC2, since in the cell lines analyzed (i) PC1 cleaves POMC into corticotropin and beta-lipotropin, (ii) PC2 cleaves POMC into beta-endorphin, an N-terminally extended corticotropin containing the joining peptide, and either alpha MSH or desacetyl-alpha MSH, and (iii) PC2 cleaves POMC at the five pairs of basic residues analyzed, whereas PC1 cleaves two of them preferentially, suggesting that PC2 has a broader spectrum of activity than PC1. These data are consistent with our hypothesis on the physiological role of PC1 and PC2 as distinct proprotein convertases acting alone or together to produce a set of tissue-specific maturation products in the brain and in peripheral tissues.     

Acetylation of melanocyte-stimulating hormone and beta-endorphin in the pars intermedia of the perinatal pituitary gland in the mouse

This report concerns ontogenetic aspects of the production and in vitro release of NH2-terminally acetylated forms of melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin by the pars intermedia of the pituitary gland of the mouse. In vitro biosynthetic analysis and radioimmunoassay revealed that approximately 12 h before birth most of the MSH in the fetal pars intermedia is present as des-N alpha-acetyl alpha-MSH. The same non-acetylated peptide is at this stage also the major release form of melanotropin. In 1-day-old mice the level of alpha-MSH and diacetylated alpha-MSH had increased considerably, although des-N alpha-acetyl alpha-MSH remained the major form. Five days after birth alpha-MSH and its diacetylated form constitute the major tissue and release form of the peptide, a situation very similar to that in adult mice. Acetylation of beta-endorphin appeared to occur earlier in development, N alpha-acetyl beta-endorphin (1-31) being the major form of endorphin already in the fetal pars intermedia. It is concluded that in the mouse acetylation of melanotropin and acetylation of beta-endorphin are not necessarily concomitant events. It could be established that the ability of the pars intermedia cells for cleaving N alpha-acetyl beta-endorphin (1-31) to yield C-terminally shortened forms of beta-endorphin develops after birth.     

Immunocytochemical location of pituitary cells containing ACTH, alpha-MSH, and beta-endorphin in Acipenser transmontanus, Lepisosteus spatula, and Amia calva

This immunocytochemical study of the pituitaries of the primitive actinopterygians, Acipenser transmontanus, Lepisosteus spatula, and Amia calva, showed a strict delineation between the hormonal fragments of proopiomelanocorticotropin (POMC) produced by corticotropes of the pars distalis and the melanotropes of the pars intermedia. Corticotropes were immunoreactive only for ACTH and not to either of the further degradation products, alpha-MSH or beta-endorphin. Melanotropes were shown to be immunoreactive to all three antisera but it is argued that immunoreactivity of melanotropes to ACTH antiserum is due to that antiserum's cross-reactivity with the cleavage product corticotropin-like intermediate peptide. The PAS positivity of both the corticotropes and the melanotropes of all three primitive fish argues for an ancient origin of a carbohydrate component of POMC and for its loss or reduction in teleosts where these cells are PAS negative.     

Evidence that glycosylation of pro-opiocortin and ACTH influences their proteolysis by trypsin and blood proteases

The role of the carbohydrate in the stabilizaion and protection of the glycoprotein, pro-opiocortin, from non-specific proteolysis by trypsin and blood proteases was studied in vitro. [3H]Arginine-labeled, glycosylated and non-glycosylated forms of pro-opiocortin were isolated from frog neurointermediate lobes and subjected to proteolysis by trypsin. The non-glycosylated form was degraded by trypsin more rapidly than the glycosylated form. Analysis of the tryptic products after trypsin treatment, showed that the non-glycosylated pro-opiocortin was cleaved to unidentified peptides within 1 min, whereas the glycosylated prohormone yielded 2 products, mol. wt. 23 000 ACTH and mol. wt. 21 000 ACTH, synthesized by the intact neurointermediate lobe. These data provide direct evidence in support of the hypothesis, derived from studies on the intact lobe (Loh and Gainer, 1978, 1979) that the glycosylation of pro-opiocortin is important: (1) to protect it against non-specific proteolysis in situ, and (2) to direct processing by limiting proteolysis. In addition, we demonstrate that glycosylated forms of ACTH are much more stable in blood than non-glycosylated forms.     

Detection and partial characterization of proopiomelanocortin-related end-products from the pars intermedia of the toad, Bombina orientalis.

Steady-state analyses were performed on the proopiomelanocortin (POMC)-related end-products present in acid extracts of the pars intermedia of the anuran amphibian, Bombina orientalis. Sephadex G-75 gel filtration chromatography indicated that immunoreactive alpha-MSH-sized material and N-acetylated beta-endorphin-related material are the major POMC-related products present in this tissue. The alpha-MSH-sized immunoreactivity was further fractionated by reversed phase HPLC. The major peak of immunoreactivity isolated by this procedure eluted with the same retention time as synthetic ACTH(1-13)amide. Cation exchange chromatography supported the conclusion that the major storage form of alpha-MSH in the pars intermedia of Bombina is ACTH(1-13)amide. Analysis of Bombina pars intermedia in culture indicated that mono-acetylated and di-acetylated alpha-MSH were the major forms of alpha-MSH secreted into the medium. The major peak of N-acetylated beta-endorphin-related material was further analyzed by cation exchange chromatography and Sephadex G-25 gel filtration column chromatography. The major storage form of beta-endorphin in this tissue is N-acetylated, has a net positive charge at pH 2.75 of +1, and has an apparent molecular weight of 1.2K. The beta-endorphin present in the pars intermedia of this tissue does not undergo further N-acetylation at the time of secretion. These results indicate that in the pars intermedia of the archaeobatrachian, Bombina orientalis, the N-acetylation of alpha-MSH is a cosecretory processing event, whereas N-acetylation of beta-endorphin is a post-translational processing event. These results are compared to other archaeobatrachian and neobatrachian pituitary POMC systems that have been analyzed.     

Functional melanocortin-2 receptors are expressed by mouse aorta-derived mesenchymal progenitor cells

A local melanocortin system is active during tissue injury and inflammation. Thus far this system has been described as autocrine in nature where local production of pro-opiomelanocortin (POMC) peptides by leukocytes feeds back on melanocortin receptor (MC-R) expressing immune cells to quell inflammatory cytokine production. Here we present evidence that POMC peptides may generate extracellular matrix (ECM) changes by inducing matrix production by cells of the mesenchymal lineage through activation of the MC2-R. Using immunoblot, we determined that mouse aorta-derived mesenchymal progenitor cells express both MC2-R and MC3-R. These progenitors respond to treatment with ACTH by increasing collagen matrix synthesis as assessed by picrosirius red stain and (3)H-proline incorporation. ACTH also induces transient increases in intracellular calcium ([Ca(2+)](i)) as assessed using the fluorescent Ca(2+) indicator, fura-2. The ACTH-induced changes in [Ca(2+)](i) are consistent with MC2-R signaling and consist of both an intracellular release and an extracellular influx of Ca(2+). Both mouse aortic mesenchymal progenitors and mouse macrophage cells express POMC and the prohormone convertase 1/3 (PC1/3) indicating they have the potential to contribute to the local production of POMC peptides. These data demonstrate functional MC2-R expression in mouse aorta-derived mesenchymal progenitors and implicate both macrophage and mesenchymal cells as relevant sources of local POMC peptides.