Biological and physical approaches on the role of piplartine (piperlongumine) in cancer.

Chronic inflammation provides a favorable microenvironment for tumorigenesis, which opens opportunities for targeting cancer development and progression. Piplartine (PL) is a biologically active alkaloid from long peppers that exhibits anti-inflammatory and antitumor activity. In the present study, we investigated the physical and chemical interactions of PL with anti-inflammatory compounds and their effects on cell proliferation and migration and on the gene expression of inflammatory mediators. Molecular docking data and physicochemical analysis suggested that PL shows potential interactions with a peptide of annexin A1 (ANXA1), an endogenous anti-inflammatory mediator with therapeutic potential in cancer. Treatment of neoplastic cells with PL alone or with annexin A1 mimic peptide reduced cell proliferation and viability and modulated the expression of MCP-1 chemokine, IL-8 cytokine and genes involved in inflammatory processes. The results also suggested an inhibitory effect of PL on tubulin expression. In addition, PL apparently had no influence on cell migration and invasion at the concentration tested. Considering the role of inflammation in the context of promoting tumor initiation, the present study shows the potential of piplartine as a therapeutic immunomodulator for cancer prevention and progression.

Web Services for Molecular Docking Simulations.

Docking process is one of the most significant activities for the analysis of protein-protein or protein-ligand complexes. These tools have become of unique importance when allocated in web services, collaborating scientifically with several areas of knowledge in an interdisciplinary way. Among the several web services dedicated to carrying out molecular docking simulations, we selected the DockThor web service. To illustrate the application of DockThor to protein-ligand docking simulations, we analyzed the docking of a ligand against the structure of epidermal growth factor receptor, an essential molecular marker in cancer research.

Ovarian and steroidal influences on neuroendocrine aging processes in female rodents.

Some Mammalian aging processes involve effects of steroids on the brain and pituitary. An ovary-dependent, neuroendocrine aging syndrome of laboratory rats and mice is described in this article. This syndrome can be attenuated during aging by chronic ovariectomy and can be prematurely induced in young rodents by sustained exposure to estradiol (E2). The limited follicular stock in the ovary is proposed to be a major pacemaker of aging in this neuroendocrine syndrome; ovarian aging may interact with neuroendocrine aging. Ovary-independent neuroendocrine changes occur as well. We also discuss developmental influences on adult aging in rodents and other examples in which adult lower mammals are sensitive to long lasting effects of steroids on the brain and pituitary. Possible molecular mechanisms are considered. In view of the long lasting effects of E2 and other steroids on lower mammals, the potential for long term effects of ovarian steroids on the human brain and pituitary warrants continued evaluation.

Molecular models of NS3 protease variants of the Hepatitis C virus.

BACKGROUND: Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed. RESULTS: The atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related beta-alpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures. CONCLUSIONS: This project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure-based drug design of a new generation of NS3 protease variants inhibitors. All models in the database are publicly accessible via our interactive website, providing us with large amount of structural models for use in protein-ligand docking analysis.

Age-related changes in proopiomelanocortin messenger ribonucleic acid levels in hypothalamus and pituitary of female C57BL/6J mice.

Concentrations of POMC-derived neuropeptides are reduced in hypothalami of aged rodents, whereas levels of POMC products in the pituitary are usually either unchanged or increased. Whether these changes reflect altered synthesis or processing of POMC or altered expression of the POMC gene has not been established. We, therefore, measured POMC mRNA in hypothalami and pituitaries of young (7-month-old), middle-aged (15-month-old), and old (31-month-old) female mice, using slot blot hybridization of total RNA to riboprobes synthesized from cloned POMC DNA fragments. Concentrations of poly(A) and ribosomal RNA were also determined, using probes synthesized from poly(T) and a cloned ribosomal DNA fragment, respectively. Hypothalamic POMC mRNA content and concentration were 30% lower in old than in young and middle-aged mice (P less than 0.01). This change was not due to a general decline in hypothalamic mRNA or rRNA levels, neither of which changed with age. In contrast to the hypothalamus, the relative concentration of POMC mRNA in the pituitary nearly doubled in old mice (P less than 0.01). This increase was secondary to a decrease in other RNA species, however, since the total pituitary content of POMC mRNA did not differ between young and old mice. These results indicate that the regulation of POMC gene expression during aging differs in hypothalamus and pituitary, and that reduced levels of hypothalamic POMC mRNA may account for the previously reported reductions in concentrations of hypothalamic POMC peptides during aging.

Tissue differences in estrogen receptor dynamics: nuclear retention, rate of replenishment, and transient receptor loss vary in hypothalamus, pituitary, and uterus of C57BL/6J mice.

Little is known about tissue differences in estrogen receptor (ER) dynamics, despite evidence that they could play a role in the tissue specificity of estrogen action. This study was designed to test the hypothesis that ER dynamics differ in uterus (UT), pituitary (PIT), and hypothalamus (HYPO), as measured by 1) duration of peak nuclear ER (ERn), 2) rate of replenishment of cytosolic ER (ERc), and 3) loss of total ER (ERt) after a bolus of estradiol (E2). Young adult mice were studied at two hormonally distinct stages of the cycle [days 2 or 3 (D2-3) and D5 (D1 = proestrus)]. Animals were injected with a dose of E2 (0.05 microgram/10 g BW) sufficient to achieve maximal ERn or with vehicle only, and ER was determined in nuclear and cytosolic fractions 1, 2, 4, 8, 12, and 24 h later. ERn peaked concomitantly with plasma E2 at 1 h in all tissues, but the duration of peak ERn varied among tissues: 4 h in HYPO compared to 1-2 h in UT and PIT. ERc replenishment was complete by 12 h in HYPO, but not until 24 h or more in PIT; replenishment in UT was intermediate (12-24 h). The transient loss of ERt after E2 injection was pronounced in UT and PIT, but was undetectable in HYPO. These tissue differences were maintained across cycle state, despite effects of cycle state on ER dynamics. The effects of cycle state on ER dynamics were also tissue specific; they were greatest in UT and absent in HYPO. On D2-3 in UT, ERn and ERt were lower, and replenishment of ERc was slower than on D5. Parallel effects of cycle state were seen in PIT, with the exception of ERn, which was unaffected. Because altered ER dynamics similar to those observed on D2-3 can be produced by progesterone pretreatment, the altered ER dynamics on D2-3 may be a consequence of recent exposure on D1 to the ovulatory surge of progesterone. Taken together, these results indicate that the mechanisms governing intracellular ER dynamics vary markedly among tissues and provide an impetus for further examination of their role in the tissue specificity of estrogen action.

Up-regulation of the uterine estrogen receptor and its messenger ribonucleic acid during the mouse estrous cycle: the role of estradiol.

Uterine estrogen receptor (ER) and ER mRNA were measured in cycling and ovariectomized (OVX) estrogen-treated mice to probe the physiological regulation of the intracellular distribution and biosynthesis of ER. On proestrus, when plasma estradiol (E2) levels are highest, the cell nuclear ER concentration was 2.4-fold greater than on metestrus. This increase was primarily attributable to an increase in total cellular ER (cytosolic plus nuclear ER) and only secondarily to an activation of ER, as measured by its redistribution from the cytosolic (i.e. nuclear-extractable) to the nuclear (nonextractable) fraction. Total cellular ER concentration was 1.8-fold higher on proestrus than on metestrus, whereas the fraction of total ER in the nuclear compartment (i.e. the percentage activated) was only 1.3-fold higher. The concentration of cellular ER mRNA was 3-fold greater on proestrus than on the other days of the estrous cycle, suggesting that the increased concentration of ER on proestrus was a consequence of increased ER gene expression. In OVX mice, physiological and, to a lesser extent, supraphysiological levels of E2 increased cell nuclear ER. As in proestrous mice, the increased ER content contributed more than ER activation to the increased cell nuclear ER concentration. Physiological, but not supraphysiological, concentrations of E2 increased ER mRNA in OVX mice. Together, these results suggest that up-regulation by E2 of ER mRNA and ER accounts for most of the increased nuclear binding of ER on proestrus. E2-dependent activation and consequent DNA binding of ER presumably initiate this process, but quantitatively account for only a small fraction of the increased nuclear binding of ER.

Differential contributions of ovarian and extraovarian factors to age-related reductions in plasma estradiol and progesterone during the estrous cycle of C57BL/6J mice.

The relative contributions of ovarian and extra-ovarian factors to the altered ovarian steroidal profiles of middle-aged mice were assessed by reciprocal, heterochronic ovarian grafting. Ovaries from cycling, young (2 months), and middle-aged (12 months) mice were exchanged by grafting under the renal capsules. Blood samples were obtained daily at midday throughout the estrous cycle for measurement of estradiol (E2) and 3-4 h after lights-out on proestrus to measure the preovulatory elevation of progesterone (P4). Middle-aged intact mice had lower mean concentrations of E2 during the cycle, no detectable midday preovulatory elevation of E2, and an attenuated preovulatory increase of P4 compared to young mice. Ovarian grafts from young donors failed to increase mean E2 levels of middle-aged mice, but did restore the preovulatory elevation of E2 and preovulatory P4 to levels of young controls. Reciprocal grafting confirmed these findings: ovaries from middle-aged donors in young hosts produced mean E2 levels equivalent to those of young mice but were unable to support a preovulatory increase of E2 or a preovulatory P4 level equivalent to that of young controls. These results reveal differential contributions of ovarian and extra-ovarian factors to age changes in E2 and P4. They indicate that ovarian aging plays an important role in attenuating the preovulatory increase of E2 and P4, but extra-ovarian, presumably neuroendocrine, age changes underlie the mean reduction of E2 levels across the estrous cycle.

The role of ACTH and adrenal glucocorticoids in the salt appetite of wild rabbits (Oryctolagus cuniculus (L)).

The selective appetites of wild rabbits for 500 mEq/1 solutions of NaC1, KC1, MgC1(2), and CaC1(2) were studied in intact and adrenalectomized rabbits during daily treatment with either 4 IU long acting ACTH, 1.0 or 2.5 mg cortisol acetate, or 2.5 mg corticosterone. The animals were individually caged and external sodium balances performed. In intact rabbits, cortisol or corticosterone produced a significant stimulation of NaC1 appetite. The response to concurrent dosage of cortisol and corticosterone was less than half of that obtained with ACTH which produced a comparable alteration of blood glucocorticoid levels but a 10-fold increase in NaC1 intake. CaC1(2) intake was increased in intact rabbits by cortisol treatment but not by corticosterone or ACTH. Adrenalectomized rabbits maintained on daily steroid replacement therapy of 0.1 mg deoxycorticosterone acetate and 0.75 mg cortisone acetate showed a normal pattern of electolyte, food, and water intake. Under these conditions ACTH produced a 4-fold increase in NaC1 intake. Further addition of cortisol and corticosterone to steroid replacement therapy produced an increase in NaC1 intake comparable to their effect on normal rabbits. Thereupon supplementation with ACTH resulted in an increase to a level at least as great as that found in ACTH treated, normal rabbits. The effects of ACTH and glucocorticoids on NaC1 appetite were synergistic. Sodium balance showed that increases in NaC1 intake were not the result of the treatment initially producing a body sodium deficit, which was then corrected by increased intake. The results provide further evidence for the hypothesis that NaC1 appetite may be hormonally regulated, and demonstrate that ACTH is capable of stimulating NaC1 intake by a previously unsuspected non-adrenal pathway.

Follicular depletion during the menopausal transition: evidence for accelerated loss and ultimate exhaustion.

Although the menopause is generally considered to be the consequence of follicular exhaustion, the relationship between follicle number and the menopausal transition has not been explicity studied. We addressed this question in 17 women, aged 45-55 yr, who were undergoing elective total abdominal hysterectomy and salpingo-oophorectomy. The women were divided into 3 groups according to their menstrual history: 1) menstruating regularly (n = 6), 2) perimenopausal (irregular menses; n = 7), and 3) postmenopausal (greater than 1 yr since last menses; n = 4). The mean ages of the 3 groups were similar. Menstrual histories were confirmed by plasma hormone levels and endometrial histology. One ovary from each woman was serially sectioned for determination of follicle numbers. The mean number of primordial follicles in the ovaries of women who were still menstruating regularly was 10-fold higher than that in perimenopausal women [1392 +/- 355 (+/- SEM) vs. 142 +/- 72]. Follicles were virtually absent in the postmenopausal ovaries. Comparison of these data with those obtained by others in younger women suggests that follicular depletion accelerates dramatically in the last decade of menstrual life. These results support the view that declining follicular reserve is the immediate cause of both the perimenopausal and menopausal transitions, and indicate that the rate and, therefore, the regulation of follicular depletion change during the final phase of reproductive life.

Aging and long-term ovariectomy alter the cytoarchitecture of the hypothalamic-preoptic area of the C57BL/6J mouse.

Long-term ovariectomy attenuates several neuroendocrine-dependent changes in reproductive function of aging female rodents, but the sites affected and underlying mechanisms are not known. This study was designed to identify effects of aging on hypothalamic-preoptic neurocytoarchitecture and to determine whether long-term ovariectomy attenuated any of those changes. Coronal sections from midportions of the preoptic area (POA), suprachiasmatic nucleus (SCN) and arcuate nucleus (ARC) were examined by quantitative light microscopy in young (5 month) and old (18-20 months) intact mice and in old mice which had been ovariectomized at 6 months of age. Neuronal density in old intact mice decreased in both ARC and POA (30%; p less than 0.05), but no significant decrease occurred in SCN. Total perikaryal area decreased only in ARC (10%; p less than 0.05), while area of perikaryal nuclei did not change in any region. Area of nucleoli increased (13%; p less than 0.005) in all three regions of old intact and old ovariectomized mice. Neither density nor size of glia changed with age. Density of blood vessels increased strikingly in ARC (150%; p less than 0.05), and in POA (30%; p less than 0.05), but not significantly in SCN of old mice. Long-term ovariectomy did not prevent any of these changes; indeed, it promoted the decline of neuronal density in SCN (40%; p less than 0.05) and neuronal area (15%; p less than 0.05) in POA. Reductions in neuronal density were not explained by expansion of the SCN or peri-ARC or peri-POA regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Loss of LH-RH neurons in the rostral forebrain of old female C57BL/6J mice.

This study reports the quantitative immunohistochemical distribution of luteinizing hormone-releasing hormone (LH-RH) neurons within the rostral forebrain of young (5-6 month) and old (26-28 month) C57BL/6J mouse. Old mice demonstrated a significant (18%) loss of LH-RH-containing neurons (p less than 0.001, ANOVA). The most striking losses involved the preoptic area (24%) and more rostral regions (26%). The presence of pituitary or mesenteric tumors in older mice did not affect the density of LH-RH neurons. These observations indicate that LH-RH neurons comprise part of the neuronal population previously reported to be reduced in the preoptic area of older mice (4).

Food restriction delays the age-related increase in GFAP mRNA in rat hypothalamus.

Astrogliosis with advancing age is correlated with increased expression of glial fibrillary acidic protein (GFAP). Hypothalamic GFAP mRNA prevalence was determined in male F344 rats of different ages that were fed ad lib (AL) and compared with that of rats that were food-restricted (FR) to 60% of AL levels. Hypothalamic GFAP mRNA increased 3-fold at 24 to 25 months in AL rats compared with 3 and 6 month groups. There were no differences in GFAP mRNA levels between AL and FR rats from 3 to 18 months. However, GFAP mRNA was significantly lower in FR than in AL rats at 24 to 25 months; FR rats reached the level of GFAP mRNA in 24 to 25 months AL rats by 33 months. Hypothalamic glutamine synthetase mRNA also increased with age in both dietary groups but did not differ between dietary groups at any age. The observation that FR delays the increased expression of GFAP in the hypothalamus during aging lends support to the hypothesis that upregulation of GFAP mRNA is a biomarker of brain aging.

Loss during aging of beta-endorphinergic neurons in the hypothalamus of female C57BL/6J mice.

Beta-endorphin (B-EP) content is often reduced in hypothalami of aging rodents. The objective of this study was to determine whether reduced B-EP content is associated with a reduced number of B-EP immunoreactive neurons. Serial coronal sections extending from the caudal hypothalamus through the retrochiasmatic area were examined by quantitative light microscopy in mature (5-6 month) and senescent (24-28 month) mice that had been ovariectomized 1 week earlier and injected with colchicine 24-48 h before sacrifice. Old mice were acyclic. As expected, B-EP immunoreactive cell bodies were restricted to the region of the arcuate nucleus. There was a 35% loss of B-EP immunopositive neurons in old, macroscopically disease-free animals. By contrast, some old animals with pituitary tumors had no loss of B-EP neurons. These results suggest that a subpopulation of B-EP neurons either die or stop synthesizing detectable concentrations of B-EP in aged mice. The basis for the absence of reduced B-EP neurons in some mice with pituitary tumors is unclear, but this observation underscores the importance of distinguishing age-related changes associated with diseases of aging from those that are independent of such diseases.

Neuroendocrine involvement in aging: evidence from studies of reproductive aging and caloric restriction.

Neuroendocrine changes contribute to female reproductive aging, but changes in other tissues also play a role. In C57BL/6J mice, neuroendocrine changes contribute to estrous cycle lengthening and reduced plasma estradiol levels, but the midlife loss of cyclicity is mainly due to ovarian failure. Hypothalamic estrogen receptor dynamics and estrogenic modulation of gene expression are altered in middle-aged cycling mice. Although insufficient to arrest cyclicity, these neuroendocrine changes may contribute to other reproductive aging phenomena, such as altered gonadotropin secretion and lengthened estrous cycles. In women, the loss of ovarian oocytes, the cause of menopause, accelerates in the decade before menopause. Accelerated oocyte loss may in turn be caused by a selective elevation of plasma follicle stimulating hormone, and neuroendocrine involvement may thus be implicated in menopausal oocyte loss. Chronic calorie restriction retards both neural and ovarian reproductive aging processes, as well as age-related change in many other physiological systems. The diverse effects of food restriction raises the possibility of an underlying coordinated regulatory response of the organism to reduced caloric intake, possibly effected through alterations of neural and/or endocrine signalling. We are therefore attempting to identify neuroendocrine changes that may coordinate the life prolonging response of animals to food restriction. Our initial focus is on the glucocorticoid system. Food restricted rats exhibit daily periods of hyperadrenocorticism, manifest as elevated free corticosterone during the diurnal peak. We hypothesize that this hyperadrenocortical state potentiates cellular and organismic homeostasis throughout life in a manner similar to that achieved during acute stress, thereby retarding aging processes and extending life span.

Dissociation kinetics of the uterine estrogen receptor-estradiol complex are unaltered in aging C57BL/6 mice.

Several, though not all, estrogen-dependent phenomena show reduced responsiveness to estradiol (E2) during aging. One factor contributing to this reduced sensitivity could be an increase in the dissociation rate of the estrogen receptor-hormone complex. We therefore studied the effect of aging on the dissociation rate of 3HE2 from the uterine cytosolic estrogen receptor (ER) of C57BL/6 mice that had been ovariectomized 48 h earlier. Measurements were made at 28 degrees C at two concentrations of cytosol. In dilute cytosol ([ER] = 0.01 nM) the dissociation of ER-3HE2 displayed a single phase, first order profile which did not differ among young (4-6 month), middle-aged (15-18 month) and old (23-30 month) mice. In more concentrated cytosol ([ER] = 2 or 6 nM) the dissociation of ER-3HE2 displayed a biphasic first order profile that consisted of an initial rapidly dissociating phase followed by a more slowly dissociating phase. There was no effect of age on the dissociation rate constant (K) of either the rapid (K-1) or slow (K-2) phase. Shortening the time allotted for the concentrated cytosol to equilibrate with 3HE2 before measuring the dissociation rate reduced the fraction of the receptor hormone complex that dissociated in the slow phase, but, once again, the dissociation profiles did not differ between age groups. These results indicate that uterine ER dissociation kinetics remain unaltered in middle-aged and old mice and are therefore unlikely to play a role in the attenuated responsiveness to estrogen during aging.

Longitudinal studies of estrous cyclicity in C57BL/6J mice: III. Dietary modulation declines during aging.

Dietary modulation of estrous cyclicity was studied throughout the reproductive lifespan to assess the stability of age-related changes in cyclicity and to probe underlying mechanisms. Animals were fed a standard diet or an isocaloric breeder diet that differed in nutrient composition to promote fecundity. In young mice, the breeder diet more than doubled the frequency of short (4-day) cycles, and, as a result, increased the total number of cycles during the cycling lifespan by 10%. Dietary potentiation of short cycles disappeared between 7 and 9 months of age, and most subsequent age-related changes in cyclicity were resistant to dietary influence. The breeder diet had no effect on the transition from 4- to 5-day cycles, the onset of acyclicity, or on the incidence or duration of persistent vaginal cornification. It only delayed the increase of very long (greater than 5-day) cycles by 1 month. These results show that most age-related changes in cyclicity are not influenced by dietary differences that affect cyclicity in young mice, and that diminished responsiveness to dietary variation is among the earliest age-related changes in the reproductive system. In addition, the results suggest that differences in cycle frequency and, presumably, in cumulative exposure to pre-ovulatory elevations of ovarian steroids do not influence the cycling lifespan in this strain of mouse.

cDNA expression arrays reveal incomplete reversal of age-related changes in gene expression by calorie restriction.

Calorie restriction (CR) extends life span and retards many age-related cellular and molecular changes in laboratory rodents. However, neither the breadth of its effects, its underlying mechanisms, nor the limits of its action is fully understood. Expression levels of 588 genes in livers from 3- and 24-month-old ad libitum-fed (AL), and 24-month-old CR (60% of AL intake) male C57BL/6J mice (four per group) were measured. Six genes met the statistical criteria for differential expression in old AL compared to young AL mice. Only one of these age-related changes was attenuated by CR. Four additional gene products, that did not change with age in AL mice, were differentially expressed in old CR compared to old AL mice. Northern and RT-PCR analyses confirmed differential expression of four of the six candidate genes identified by the array results. Many of the identified genes have not previously been reported to be affected by CR or aging. Some of the age-related changes in gene expression are consistent with an increased vulnerability of the aged liver to carcinogenic or other insults, with only partial protection against insult by CR. Incomplete reversal by CR of age-related changes in gene expression provides a potentially important path for probing the limits of CR action. These results also show the importance of independent confirmation in expression array profiling of age-related changes in gene expression.

Evaluation of the biological properties of parathyroid hormone and analogues in a vascularly isolated parotid gland-based assay.

Infusion of bovine parathyroid hormone (bPTH) preparations into the arterial blood supply of the vascularly isolated parotid gland in anaesthetized sheep increases salivary phosphate concentration and gland blood flow rate with rapid onset and offset of action. These responses have been used as a bioassay for PTH and PTH analogues and for assessing the properties of an in-vitro inhibitory analogue [Nle-8, Nle-18, Tyr-34]bPTH-(3-34)amide. [Nle-8, Nle-18, Tyr-34]bPTH-(1-34)amide at 10(-9) to 10(-8) mol/l was four to five times more potent than bPTH(1-34) on both salivary phosphate and blood flow assays. Human PTH(1-34) was not significantly more potent than bPTH(1-34). The [Nle-8, Nle-18, Tyr-34]bPTH-(3-34)amide analogue had very slight agonist activity at 3 X 10(-7) mol/l and at a 100:1 ratio of analogue to PTH it completely inhibited the action of bPTH(1-34) on phosphate secretion and gland blood flow. It caused partial inhibition at 10:1 and had no evident effect at 1:1. These results differ from previous in-vitro results and indicate that the preparation may be valuable for evaluation of agonist and antagonist analogues of PTH. The vascularly isolated parotid gland of the sheep permits repeated random testing of analogues in a control-test-control sequence and the results indicate high sensitivity to PTH in a rapidly reactive in-vivo system with two responding parameters.

Local action of parathyroid hormone (1-34) and (1-84) at the parotid gland of sheep.

Administration of bovine parathyroid hormone (PTH) preparations increased the phosphate concentration in the parotid saliva of sheep. Data on the site of action of PTH (1-84) were obtained by (a) equimolar infusions of PTH (1-84) and (1-34) directly into the arterial blood supply of the vascularly isolated parotid gland in anaesthetized sheep, (b) intravenous infusion of PTH (1-84) at a similar rate and (c) intra-arterial infusion of PTH (1-84) with complete drainage of the venous effluent from the gland during the infusion. Results showed substantial time- and dose-response identity of the two peptides, at 10(-9) to 4 X 10(-9) mol/l in arterial blood, in raising salivary phosphate concentration. The effect of PTH (1-84) was not due to recirculated fragments because the response was obtained when recirculation was prevented by complete venous drainage and little or no response occurred when the same infusion was given i.v.