Specific inhibition of insulin receptor dephosphorylation by a synthetic dodecapeptide containing sulfotyrosyl residues as phosphotyrosyl mimetic.

We have synthesized a tris-sulfotyrosyl dodecapeptide (3S-peptide-I) that corresponds to the major autophosphorylation domain within the insulin receptor beta-subunit and showed that it potently inhibited insulin receptor dephosphorylation by protein tyrosine phosphatases (PTPases) in vitro. 3S-peptide-I also inhibited tyrosine dephosphorylation of a synthetic peptide by the recombinant PTPase PTP-1B, indicating that 3S-peptide-I interacts directly with PTPase, causing its inactivation. The peptide had no effect on the activity of serine/threonine phosphatases, PP-1 and PP-2A, or alkaline phosphatase. Furthermore, we found that the introduction of a N-stearyl derivative of 3S-peptide-I in CHO/HIRc cells caused a significant increase in insulin-stimulated phosphorylation of the insulin receptor. In contrast, ligand-stimulated phosphorylation of epidermal growth factor (EGF) receptor in CHO cells overexpressing EGF receptors was not affected by the presence of N-stearyl-3S-peptide-I. These data suggest that by inhibiting dephosphorylation of the insulin receptor in intact cells, 3S-peptide-I may specifically enhance insulin signalling.

A synthetic peptide derived from a COOH-terminal domain of the insulin receptor specifically enhances insulin receptor signaling.

The role of the insulin receptor COOH-terminal domain in the regulation of insulin signal transduction was explored with a variety of synthetic peptides. One of the peptides, termed peptide HC, whose structure corresponds to residues 1293-1307 of the insulin proreceptor sequence, enhanced insulin-stimulated autophosphorylation of the insulin receptor in cell-free systems and in semipermeabilized Chinese hamster ovary (CHO) cells that had been transfected with an expression plasmid encoding the human insulin receptor (CHO/HIRc) at concentrations where there was no detectable effect on basal autophosphorylation levels or on receptor dephosphorylation. A lipophilic analogue of peptide HC, stearyl peptide HC, added to intact CHO/HIRc cells enhanced significantly insulin-stimulated insulin receptor autophosphorylation while having no effect on ligand-stimulated receptor phosphorylation in CHO cells overexpressing either the IGF-1 receptor or epidermal growth factor receptor. Addition of stearyl peptide HC to CHO/HIRc cells resulted in a 2.4 +/- 0.3-fold increase in the amount of insulin-stimulated phosphatidylinositol 3-kinase detected in anti-IRS-1 immunoprecipitates and a 2.1 +/- 0.6-fold increase in the levels of tyrosine phosphorylation of mitogen-activated protein kinase in response to insulin. Finally, a derivative of peptide HC coupled to a biotin moiety was prepared and showed to bind with the beta-subunit of the wild-type insulin receptor and a truncated receptor that lacks 43 amino acids from its carboxyl terminus. However, there was little binding, if any, of the peptide with the IGF-1 receptors or the epidermal growth factor receptors. Taken together, our data demonstrate that a pentadecapeptide related to the carboxyl terminus of the insulin receptor binds to the insulin receptor beta-subunit and that this interaction may contribute to the increased receptor's intrinsic activity and signal transduction.

Age-dependent reduction in insulin secretion and insulin mRNA in isolated islets from rats.

Aging is an etiologic factor in non-insulin-dependent diabetes mellitus. To characterize the beta-cell abnormalities that occur with age, we investigated glucose-stimulated insulin release, pancreatic insulin content, and mRNA levels for islet-specific genes in aging Wistar rats. Ten minutes after glucose stimulation, 6-mo-old islets had approximately 40% more cells secreting insulin than 24-mo-old islets (P < 0.0001); after 1 h, 67 +/- 1.0% islets from 6-mo-old rats secreted insulin, compared with 51 +/- 3.5% from 24-mo-old rats (P < 0.0001). The amount of insulin secreted by each beta-cell was also less in the older animals (P < 0.0001). Despite increases in islet size (P < 0.0001) and beta-cell number (P < 0.0001) with age, whole pancreas insulin content showed that 24-mo-old pancreas had less insulin than 6-mo-old pancreas (0.61 +/- 0.06 vs. 0.84 +/- 0.08 microgram/mg pancreatic protein; P < 0.05). Finally, insulin mRNA levels declined to 50% of the newborn value in 24-mo-old islets (P < 0.0001), whereas glucagon mRNA levels showed a very modest decline with age. Somatostatin mRNA levels did not vary significantly. In summary, it appears that in Wistar rats there is a progressive decline in beta-cell activity with age. This decline may represent the biological features of the age-dependent risk of developing diabetes.

A synthetic tris-sulfotyrosyl dodecapeptide analogue of the insulin receptor 1146-kinase domain inhibits tyrosine dephosphorylation of the insulin receptor in situ.

A synthetic tris-sulfotyrosyl dodecapeptide (TRDIY(S)ETDY(S)Y(S)RK-amide), whose primary sequence is identical to the 1142-1153 sequence of the insulin proreceptor, inhibited insulin receptor dephosphorylation in solubilized membranes, and digitonin-permeabilized cells derived from Chinese hamster ovary (CHO) cells expressing high levels of human insulin receptors (CHO/HIRc). It also inhibited the dephosphorylation of a synthetic tyrosine phosphorylated substrate by recombinant PTP-1B, a protein tyrosine phosphatase (PTPase), indicating that it acted via interaction with PTPase(s). A N-stearyl derivative of the peptide caused an approximately 4.5-fold increase in insulin-stimulated receptor autophosphorylaction in intact CHO/HIRc cells. The peptide displayed specificity toward tyrosine-class phosphatases only, as it had no effect on the activities of the serine/threonine phosphatases PP-1 and PP-2A, or alkaline phosphatase. The tyrosine sulfate ester bonds of the peptide were stable when incubated with PTP-1B (1 h, 30 degrees C). These data suggest that the sulfotyrosyl peptide functions as a nonhydrolyzable phosphotyrosyl peptide analogue capable of direct interaction with PTPase catalytic domain.

Dynamic regulation of intact and C-terminal truncated insulin receptor phosphorylation in permeabilized cells.

Using digitonin-permeabilized Chinese hamster ovary (CHO) cells that were transfected with intact human insulin receptors (CHO/HIRc cells), we examined insulin receptor phosphorylation and dephosphorylation using pulse-chase techniques. Insulin activated receptor autophosphorylation on tyrosyl residues to a level severalfold over basal, reaching maximal levels after 2, 5, and 10 min of stimulation at 34, 18, and 6 degrees C, respectively. Phosphopeptide analysis revealed that the triply phosphorylated form of the 1146-kinase domain of the insulin receptor was the major species, which is characteristic of the fully active tyrosine kinase function. The dephosphorylation reaction was time- and temperature-dependent with t1/2 values of 0.67 and 2 min at 18 and 6 degrees C, respectively. Vanadate completely inhibited dephosphorylation. Under similar permeabilization conditions when compared with CHO/HIRc cells, CHO/delta CT cells (CHO cells overexpressing a mutated form of the receptor with a 43 amino acid deletion at the C-terminus) stimulated with insulin exhibited larger increases in receptor autophosphorylation levels and in tyrosine kinase activity toward a synthetic peptide substrate; the rate of CHO/delta CT receptor dephosphorylation was not reduced. There was near-complete absence of insulin receptor substrate 1 (IRS-1) in the cell ghosts after permeabilization. We therefore examined the pattern of tyrosine phosphorylation and dephosphorylation of residual cellular proteins in permeabilized CHO/HIRc cells by Western blot analysis. In addition to the 95-kDa receptor beta-subunit, we detected the phosphorylation of two glycoproteins which included the commonly found 120-kDa protein and a novel 195-kDa protein whose dephosphorylation rate is slower than that of receptor beta-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Stress-induced alterations of immunity in hypophysectomized rats.

Stress-induced suppression of mitogen-induced lymphocyte proliferation was demonstrated in hypophysectomized rats. Stress effects on the numbers of peripheral blood lymphocytes and lymphocyte subsets and on splenic natural killer cell activity require the presence of pituitary. A pituitary-dependent restraining influence on stress-induced alteration of immunity is described. These results indicate that stress-induced modulation of immunity is complex and includes a range of enhancing and inhibitory mechanisms.

Isolation and characterization of a corticotropin-releasing hormone-like peptide from human placenta.

Immunoreactive CRH was detected in extracts of human term placentae [5.2 +/- 0.8 (+/- SE) pmol/g wet wt; n = 9]. Molecular sieve chromatography revealed three size classes of immunoreactive CRH. The major species eluted with the Kav of synthetic rat CRH; the minor species had apparent mol wt (MW) of 18,000 and 8,000. A placental CRH-(1-41)-sized peptide was isolated by fractional acetone precipitation, molecular sieve chromatography, and sequential reverse phase high performance liquid chromatography steps. This peptide had the same chromatographic behavior as did rat CRH in all high performance liquid chromatographic isolation steps as well as the same UV absorbance to immunoreactive CRH ratio after the final purification step. Purified placental CRH stimulated ACTH release from anterior pituitary tissue in a dose-dependent manner and was equipotent with synthetic rat CRH. Partial sequencing indicated that 32 amino acids of this peptide are identical to those of rat and human CRH (sequence deduced from genomic sequence), and comparative peptide mapping with rat CRH provided further evidence that the placental CRH-like peptide is very homologous if not identical to CRH. The high mol wt placental CRH fractions also were partially purified by acetone precipitation, immune affinity chromatography, and gel filtration. Neither of these materials [big form (MW, 18,000) or intermediate form (MWr, 8,000)] stimulated ACTH release from rat pituitary tissue in vitro. Limited trypsin digestion of the highest MW CRH, followed by gel filtration analysis, resulted in conversion to the smaller [8,000 MW-sized and CRH-(1-41)-sized] forms. The detection of a CRH-like peptide in placenta together with our previous demonstration of plasma immunoreactive CRH in pregnant women suggest that the placenta synthesizes and secretes CRH into the maternal circulation.

Immunoreactive corticotropin-releasing hormone in human plasma during pregnancy, labor, and delivery.

We previously reported that immunoreactive corticotropin-releasing hormone (CRH) is present in human placenta and third trimester maternal plasma, and that such material is very similar to rat CRH and the predicted structure of human CRH. We suggested that maternal plasma immunoreactive CRH may be of placental origin. To further investigate this possibility, we measured plasma immunoreactive CRH in women during pregnancy, labor, and delivery and 1 and 2 h postpartum, and in nonpregnant women. Umbilical cord plasma and placental CRH concentrations were also measured. In the first trimester of pregnancy, the mean maternal plasma level was 5.9 +/- 1.0 pg (+/- SEM)/ml (n = 24), not significantly different from that in 10 nonpregnant women (5.8 +/- 0.8 pg/ml). Plasma CRH concentrations progressively increased during pregnancy (second trimester, 35.4 +/- 5.9 pg/ml (n = 39); early third trimester (28-34 weeks), 263 +/- 41 pg/ml (n = 14); late third trimester (35-40 weeks), 800 +/- 163 pg/ml (n = 20)]. There was a significant correlation between maternal plasma CRH levels and weeks of pregnancy. Plasma CRH concentrations were further elevated (2215 +/- 329 pg/ml; n = 9). During early labor, peaked at delivery (4409 +/- 591 pg/ml; n = 28), and declined rapidly after delivery [1 h postpartum, 1042 +/- (353 pg/ml (n = 13); 2 h postpartum, 346 +/- 91 pg/ml (n = 13)]. There was a significant correlation (r = 0.562; P less than 0.01) between matched maternal plasma and placental CRH concentrations. The mean umbilical cord plasma CRH level (50.6 +/- 6.1 pg/ml; n = 28) was much lower than that in the mother at the time of delivery. Umbilical venous plasma CRH levels were significantly greater than those in simultaneously obtained umbilical arterial plasma (70.8 +/- 11.3 and 41.8 +/- 4.9 pg/ml, respectively; n = 11). There was a significant correlation (r = 0.384; P less than 0.05) between maternal and fetal CRH concentrations. Gel filtration of plasma obtained from women during the third trimester, at delivery, and early postpartum and placental extracts revealed two major peaks of immunoreactive CRH: a high mol wt peak and one at the elution position of rat CRH. In contrast, only rat CRH-sized material was detected in plasma from nonpregnant women and umbilical cord plasma. Maternal plasma immunoreactive CRH-sized material stimulated ACTH release from anterior pituitary tissue in a dose-dependent manner and was equipotent with rat CRH.(ABSTRACT TRUNCATED AT 400 WORDS)

Induced expression of the glucocorticoid receptor in the rat intermediate pituitary lobe.

Synthesis and release of pro-opiomelanocortin-derived peptides are under differential regulation in the anterior and intermediate lobes of the pituitary. Glucocorticoids inhibit synthesis of pro-opiomelanocortin-related peptides in the anterior lobe but not in the intermediate lobe. These two lobes are also characterized by differences in neural innervation and blood flow, both of which may represent routes of access for regulatory factors (the intermediate lobe is avascular). Immunoreactive glucocorticoid receptor, which can be demonstrated in many tissues, is absent from the intermediate lobe. Immunocytochemistry was used to demonstrate the presence of immunoreactive glucocorticoid receptor in the intermediate lobe after pituitary stalk transection, neurointermediate lobe grafts to kidney capsule, or monolayer culture of neurointermediate pituitary cells. This appearance of the glucocorticoid receptor is presumably a consequence of removal of intermediate pituitary cells from neural influences that may be responsible for inhibiting their expression under normal conditions in vivo.

Two distinct insulin-related molecules in the guinea pig: immunological and biochemical characterization of insulin-like immunoactivity from extrapancreatic tissues of the guinea pig.

In this study we extracted guinea pig brain and testis with; the extract was adsorbed to and eluted from cartridges (the Sep-Pak C18 procedure). We found this procedure superior for recovering crystalline insulin added to buffers or tissues, and for recovering endogenous insulin from plasma, but inferior for recovery of insulin from tissues. However, we did find 'rat/pork' type-insulin in guinea pig brain and testis (5-50 pg/g wet weight tissue). Our results with the Sep-Pak C18 procedures were reproduced by four other laboratories (who found 4-60 pg/g wet weight of tissue) and similar findings were also obtained by an independent investigator. Thus, we conclude that extrapancreatic tissues of guinea pigs have a second type of insulin-related material that is more typical of other mammalian insulins, but that the amount recovered is dependent upon the extraction procedure utilized.

Immunoreactive corticotropin-releasing factor is present in human maternal plasma during the third trimester of pregnancy.

Immunoreactive (IR) corticotropin-releasing factor (CRF)-like activity was detectable in the majority of plasma samples obtained from women in the third trimester of pregnancy (68.7 +/- 23.6 pg/ml (14.4 +/- 4.9 fmol/ml); mean +/- SE, n = 15), but not in plasma (less than 10 pg/ml) from first (n = 9) or second (n = 11) trimester of pregnancy, 1 day post partum (n = 7), non-pregnant women (n = 10), or in plasma obtained from patients with Cushing's disease (n = 2) or Nelson's syndrome (n = 1), or in basal (n = 6) or ether-stressed (n = 6) rat plasma. Gel filtration of third trimester pooled plasma revealed that the majority of such material eluted with Kav of rat CRF (1-41). The IR CRF (1-41)-sized material eluted with the identical retention time as rat CRF in a reverse phase high performance liquid chromatography (HPLC) system. The detection of IR CRF exclusively in third trimester maternal plasma, together with our previous demonstration that material physicochemically indistinguishable from it is present in human term placental extracts, suggests that the placenta may be the source of plasma IR CRF.

Demonstration of immunoreactive beta-endorphin- and gamma 3-melanocyte-stimulating hormone-related peptides in the ovaries of neonatal, cyclic, and pregnant mice.

Antisera against proopiomelanocortin (POMC)-derived peptides have previously been employed to demonstrate immunostainable materials in the male reproductive tract and in the corpus luteum of rat ovary. The present study was designed to determine how the distribution of such stainable materials varies in mouse ovary as a function of the reproductive status of the animal. Peptide-like activities were localized with the unlabeled antibody peroxidase-antiperoxidase (PAP) technique in ovaries removed from mice during fetal and neonatal development, during different stages of estrous cycle, and during pregnancy, with antisera against beta-endorphin, gamma 3MSH, and an extended N-terminal portion of POMC (16 K). beta-endorphin-like activity was also quantified in ovarian extracts by RIA. Immunostainable beta-endorphin, gamma 3MSH, and 16 K fragment-like activities were present in ovaries of pregnant and normally cycling (but not immature) mice. Intense staining was found predominantly in the corpora lutea. Less intense staining was observed in the interstitium and in the following parts of large follicles: parietal granulosa, corona radiata, and cumulus oophorus. When neonatal mice were injected with hCG, immunostainable beta-endorphin-like material in the ovarian interstitial area increased. Treatment with PMSG increased staining in both secondary follicles and the interstitium. Immunoassayable beta-endorphin-like activity was twice as high (per g wet wt) at pregnancy as during the cycle. We conclude that peptides similar or identical to POMC and/or its components are present in ovarian cells and that the concentration of such material appears to be regulated by gonadotropins.

The ontogeny of immunoreactive beta-endorphin in fetal, neonatal, and pubertal testes from mouse and hamster.

UNLABELLED: Derivatives of proopiomelanocortin (POMC), physicochemically similar to beta-endorphin and desacetyl alpha MSH, have been identified in adult testes, where these peptides were localized to Leydig cells. In the present study, the presence of immunostainable derivatives of POMC was established in fetal, neonatal, and pubertal testes with the unlabeled antibody peroxidase-antiperoxidase method. Specificity of staining was established by absorption of primary antisera with excess antigen. In the mouse, immunoreactive beta-endorphin was detectable in a few primitive interstitial cells on day 14 of gestation, the day after testicular differentiation. Thereafter, the number of immunostainable cells progressively increased throughout fetal life, so that at birth, they comprised 55% of the total interstitial cells. After birth, the number of immunostaining cells declined, so that they were only 12% of interstitial cells by 5 days of age. After 10 days of age, the number of immunopositive cells progressively increased, and by 40 days, interstitial cells showed intense staining comparable to that in adult mice. At 10 days of age, when the number of immunostainable cells was low, hCG treatment increased both the number and staining intensity of beta-endorphin-positive cells to those seen in adult testes. Antibodies directed against gamma MSH, a peptide within the N-terminal segment of POMC, also produced specific staining of fetal and adult interstitial cells in the mouse. In the hamster, the pattern of staining with anti-beta-endorphin in fetal, neonatal, and pubertal interstitial cells was similar to that observed in mice; the number and staining intensity of immunostainable cells increased during fetal life, declined after birth, and rose again at puberty. IN CONCLUSION: 1) the number and staining intensity of immunostainable interstitial cells have two peaks in mouse and hamster, at birth and after puberty; 2) the number and staining intensity of mouse interstitial cells can be increased by hCG; and 3) the development of immunostainable beta-endorphin activity correlates with the previously reported spontaneous and hCG-induced maturation of morphology and enzyme activities of Leydig cells.

Demonstration of in vivo synthesis of pro-opiomelanocortin-, beta-endorphin-, and alpha-melanotropin-like species in the adult rat brain.

An in vivo labeling technique was utilized to demonstrate the in situ biosynthesis of pro-opiomelanocortin (POMC)-, beta-endorphin- and alpha-melanotropin (MSH)-like molecular species in rat brain. Unrestrained adult female rats were bilaterally cannulated in the hypothalamic arcuate nuclear region; [35S]methionine was infused either over a 15-min period with sacrifice 2 hr subsequently, or at a constant rate for 6 hr prior to sacrifice. Sequential immune-affinity chromatography and several chromatographic and electrophoretic techniques were employed to detect and characterize POMC-related material in the hypothalamic arcuate nuclear region, preoptic area, and median eminence. Four molecular species containing both corticotropin (ACTH) and beta-endorphin antigenic determinants within the same molecules were detected in the arcuate nuclear region and preoptic area. Two forms were similar to rat pituitary POMC with respect to apparent molecular weight (35,000 and 31,500) and [35S]methionine-containing tryptic fragments (one methionine each in N-terminal glycopeptide, ACTH, and beta-endorphin sequences of rat POMC). The other two forms (apparent Mr of 21,000 and 19,000) contained only the labeled tryptic fragments characteristic of ACTH and beta-endorphin. The detection of the latter forms suggests that POMC in brain, unlike its post-translational processing in rat pituitary, undergoes primary cleavage between the N-terminal peptide and the ACTH sequence. Peptides physicochemically indistinguishable from authentic beta-endorphin and des-acetyl alpha-MSH were detected in approximately equimolar amounts in all three brain regions. The ratio of POMC-like material to the alpha-MSH- and beta-endorphin-sized peptides was highest in the arcuate nuclear region, suggesting that POMC-like proteins are synthesized in the arcuate nuclear region and are processed into the smaller molecular species during axonal transport.

Identification and possible function of pro-opiomelanocortin-derived peptides in the testis.

Using antibodies against peptides derived from different portions of the POMC molecule, immunocytochemical evidence suggests that this precursor and/or the peptides present within it are localized in testicular Leydig cells of at least five species. There is no evidence for the localization of these peptides or their precursor in any other cell type in this organ. Examination of testicular extracts by gel filtration, SDS-PAGE, and RP-HPLC indicate that the testis contains low concentrations of POMC-derived peptides relative to brain. Further analysis indicates that POMC is processed to alpha-MSH and beta-endorphin similar to its processing in intermediate pituitary lobe and brain. The relative mobilities of immunoreactive alpha-MSH and beta-endorphin on RP-HPLC columns indicate that they are in the unacetylated state as in brain and in contrast to the acetylated forms in the intermediate pituitary lobe. The potential for Leydig cells to synthesize POMC and its peptides was suggested by the demonstration of POMC-like mRNA in total testis and Leydig cell cultures. The size of the POMC-like mRNA is approximately 150 base pairs shorter than anterior or intermediate pituitary POMC mRNA. POMC-like mRNA activity has also been localized to Leydig cells in sections of testes using in situ hybridization. Immunostainable beta-endorphin and other POMC-derived peptides are present in testicular Leydig cells during fetal life and following puberty at times when testosterone secretion is maximal. The accumulation of immunostainable POMC-derived peptides in Leydig cells is dramatically increased by LH and hCG. A variety of observations suggests that testicular cells can respond to POMC-derived peptides. ACTH and the MSHs stimulate growth and cAMP accumulation in Sertoli cells. By contrast, studies using antagonists suggested that beta-endorphin and/or another testicular opioid inhibit Sertoli cell proliferation and ABP secretion. These observations are consistent with the postulate that different portions of the POMC molecule may have opposite effects on Sertoli cell function and suggest a mechanism by which Leydig cells could modulate Sertoli cell activity. Intratesticular administration of opiate antagonists inhibits testosterone secretion both in vivo and in vitro. These observations suggest that Leydig cell-derived beta-endorphin may facilitate testosterone secretion either directly or indirectly. The finding of POMC and its derivative peptides in testis, ovary, adrenal, and placenta suggests that all steroid hormone-secreting organs in mammals may utilize this peptidergic system.

Characterization of immunoreactive proopiomelanocortin-related peptides in rat testes.

alpha-Endorphin, beta-endorphin, gamma-endorphin, and N-terminal ACTH immunoreactivity were detectable in acid extracts of rat testes with concentrations of 0.07 +/- 0.01, 0.18 +/- 0.03, 0.06 +/- 0.01, and 0.33 +/- 0.08 (+/- SD) pmol/g wet wt, respectively. The forms of these immunoreactive peptides were characterized by reverse phase high performance liquid chromatography. Immunoreactive beta-endorphin was also analyzed by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results indicated that the major form of immunoreactive beta-endorphin present appears to be beta-endorphin-(1-31). No alpha-N-acetylated forms of beta-endorphin or beta-lipotropin-sized material were detected. Immunoreactive alpha- and gamma-endorphin appear to be present as alpha-endorphin and des-Tyr1-gamma-endorphin, respectively. Immunoreactive alpha MSH was present as its desacetylated form. No immunoreactive ACTH fractionating with ACTH-(1-39) or its glycosylated forms was detected. This peptide profile is most similar to that seen for proopiomelanocortin-derived peptides in the brain. The low concentrations of these peptides in rat testes suggest a paracrine function.

Are messenger molecules in microbes the ancestors of the vertebrate hormones and tissue factors?

Peptides very similar to hormones and other messenger molecules of vertebrates have been detected in extracts of unicellular eukaryotes (and prokaryotes). We present arguments to suggest the possibility that these molecules 1) originated evolutionarily in unicellular microbes, 2) serve as intercellular messenger molecules in these organisms, and 3) represent the phylogenetic ancestors of the hormones and neurotransmitters as well as paracrine and other tissue factors of the vertebrates. We suggest that the biochemical elements of intercellular communication arose very early in evolution and are highly conserved; evolution largely changed the anatomy, i.e., the nature of the secretory cell, the target cell, and the fluid compartment that carries the messenger molecule from one to the other. Such an approach suggests a more rational relationship between different modes of intercellular chemical signaling in vertebrates.

Corticotropin and beta-endorphin-like materials are native to unicellular organisms.

Multiple molecular forms of immunoreactive corticotropin (ACTH) and beta-endorphin were present in extracts of a unicellular eukaryote (Tetrahymena pyriformis). One form of immunoreactive ACTH reacted similarly with two different ACTH antisera (one specific for the 11-24 sequence and the other with determinants within sequences 1-14 and 17-39) and migrated with synthetic hACTH-(1-39) in a gel filtration system. This form also exhibited ACTH bioactivity in a dispersed rat adrenal cell bioassay system, with a mean immunoassay/bioassay ratio of 1.5. Gel filtration revealed multiple size classes of immunoreactive beta-endorphin; a major peak of radioreceptor activity was detected which exhibited a K(av) similar to that of authentic beta-endorphin. A major portion of immunoreactive beta-endorphin-sized material exhibited retention times similar to those of synthetic human and camel beta-endorphin upon reverse-phase high-pressure liquid chromatography. These distinctive properties and specificities would seem to exclude the presence of limited homologies with sequences present in other proteins. High molecular weight material containing both ACTH and beta-endorphin antigenic determinants was also demonstrated, suggesting, but not proving, the presence of a common precursor molecule.