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Biomedical subjects

S Lahiri

Publications and source records attributed to S Lahiri.

At least 127 records · Page 7Linked to original sources
PubMed1992

Carotid chemoreceptor response to natural stimuli in the newborn kitten.

The activities of carotid chemoreceptors at three levels of inspired PO2 (55, 145 and 690 Torr) and at two levels of inspired PCO2 (35 and 70 Torr in O2) were studied in 28 anesthetized, mechanically ventilated kittens aged 0-17 days. A biphasic response to hypoxia was found in 46% of them: the chemosensory activity increased to a peak within 30 sec after the initial response to hypoxia and thereafter declined slowly to a stable value. The steady-state single-fiber chemosensory activity at an inspired PO2 of 55 Torr was significantly lower in kittens less than 10 days old (mean +/- SE: 5.8 +/- 0.6 impulses.sec-1) than in the older ones (8.8 +/- 1.3 impulses.sec-1, P less than 0.03). The response curves to arterial PO2 were hyperbolic in both groups but the curve for the younger kittens was displaced to the left of the curve for the older ones. The response to hypercapnia was a progressive increase in chemosensory activity with little evidence of rapid or slow adaptation. The response to hypercapnia was significantly stronger in the older kittens than in the young ones. It is concluded that, in the kitten, the carotid chemoreceptor response to hypoxia may be biphasic. The responses to hypoxia and hypercapnia are already developed but are weak at birth and continue to develop further during the first weeks of postnatal life.

Animals↗
PubMed1992

Dual responses of carotid chemosensory afferents to dopamine in the newborn kitten.

The effects of dopamine and of dopamine D2 receptor blocker haloperidol on the activity of carotid chemoreceptors were studied in 24 anesthetized, paralyzed and artificially ventilated newborn kittens aged 0-17 days. Single or few fiber preparations of chemoreceptors were made from one carotid sinus nerve. The responses of the chemosensory afferents to intravenous injections of dopamine (5-50 micrograms.kg-1) were studied in kittens breathing air and 8% O2 in N2. The effects of haloperidol on the chemosensory activity in air or 100% O2 and on the chemosensory response to hypoxia were studied. Dopamine inhibited the chemosensory discharge in 25/44 studies in normoxia. Of the 9 studies performed in hypoxia, dopamine was excitatory in 5 or had no effect in 4 (P < 0.05 vs normoxia). Inhibition of the chemosensory discharge was observed in 24/37 studies performed in kittens aged more than 3 days and was predominantly excitatory in 6/7 studies in kittens aged 0-3 days (P < 0.01). One minute after haloperidol, the chemosensory discharge had increased significantly in all experiments. The biphasic pattern of chemosensory response to hypoxia (Marchal et al., Respir. Physiol. 87: 183-193, 1992) was not changed by haloperidol. The steady-state chemosensory activity was significantly increased after haloperidol, respectively from 3.9 +/- 0.7 impulses.sec-1 to 9.8 +/- 1.2 impulses.sec-1 in air, and from 13.1 +/- 1.4 impulses.sec-1 to 17.8 +/- 2.4 impulses.sec-1 in hypoxia (mean +/- SEM, P < 0.03). It is concluded that the dopaminergic mechanisms are active in the carotid body of the kitten, and the observed responses to dopamine and haloperidol are qualitatively similar to those reported in the adult cat.

Action Potentials↗
PubMed1992

Chemosensitivity and regulation of ventilation during sleep at high altitudes.

The paper presents new observations on young high altitude natives (Andes and Himalayas), testing the hypothesis that periodic breathing with apnea during sleep is determined by their ventilatory sensitivity to hypoxia and its interaction with the sleep state. The hypothesis is in general supported by the evidence. But, contrary to expectation, the ventilatory sensitivity to hypoxia in the Sherpa children was significantly lower than those in the Andes. Despite that departure, the magnitude of ventilatory periodicity among the subjects was internally consistent with their ventilatory sensitivity to hypoxia. Although the carotid chemosensory input is the pacesetter for the reflexive periodicity, mechanisms in the central nervous system can influence it significantly.

Adolescent↗
PubMed1992

Thyrotropin modulation of epidermal growth factor (EGF) binding to receptors on cultured thyroid cells.

Previous studies had shown that epidermal growth factor (EGF) will stimulate growth of cultured thyroid cells in vitro, and TSH will stimulate total assayable EGF receptor in cultured porcine thyroid cells. In this study, we report the effect of TSH on EGF binding to human thyroid cells. Addition of bTSH (1 mU/mL) in binding buffer during receptor assay stimulated specific EGF binding to cells, with an increase of 44% observed over the control after 1 h incubation at 37 degrees C. Affinity crosslinking of the [125I]EGF-receptor complex showed a single labeled band with molecular size of 170 kD. No additional band was detected in the presence of TSH. Preincubation of cells with chloroquine, which inhibits lysosomal degradative enzyme activity, caused a continuous accumulation of bound EGF over a 4 h study period at 37 degrees C, and TSH stimulated an increase in internalized EGF. In the presence of chloroquine, total specific bound EGF was linearly correlated to incubation time up to 4 h and can be expressed as Bound = slope*time+intercept (time0) Addition of TSH during the binding assay significantly increased the value of the slope when compared to control (p < 0.002). The rate at which prebound [125I]EGF was released into medium was not affected by the presence of TSH, indicating that TSH-enhanced binding may not be attributed to a reduction in EGF degradation. Coincubation of thyroid cells with EGF at 0 and 1 ng/mL and increasing concentrations of TSH (0-10 mU/mL) indicated that EGF stimulated thymidine incorporation, although TSH failed to synergistically enhance EGF-stimulated cell growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed1992

Intracellular pH and oxygen chemoreception in the cat carotid body in vitro.

To test the hypothesis that O2 chemoreception in the carotid body (CB) is mediated by cellular acidosis, we simultaneously measured responses of the chemosensory and intracellular pH (pHi) to agents that are known to change pHi and studied the effects of hypoxia and ischemia on these variables in the cat CB. The CB was perfused and superfused in vitro with a modified Tyrode's solution at 36.0 +/- 0.5 degrees C with or without CO2-HCO3- (pH 7.40) and equilibrated at a given PO2. Chemosensory discharges were recorded from the whole carotid sinus nerve. To measure pHi changes, the CB was loaded with the pH-sensitive indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and the fluorescence (excitation 420-490 nm, emission greater than 515 nm) was detected by an intensified charged coupled device camera with an epifluorescence macroscope. Boluses of Tyrode's solution (0.5 ml, free of CO2-HCO3-) containing sodium acetate or NH4Cl prolonged perfusion of acid Tyrode's solution (pH 7.20-6.50), and boluses of Tyrode's solution with CO2-HCO3- were used. A decrease of fluorescence indicated pHi turning acid, and an increase of fluorescence indicated a change in alkaline pHi. Chemosensory activity varied inversely with the fluorescence change after application of these agents. Interruption of perfusate flow or application of hypoxic perfusate resulted in large increases in chemosensory discharge without any change in the fluorescence. The results indicated that chemosensory responses to brief ischemia and hypoxia were not mediated by a fall of pHi of CB cells, whereas those to CO2 and extracellular acidity were associated with decreases in pHi.

Animals↗
PubMed1992

Receptors for endothelin in cultured human thyroid cells and inhibition by endothelin of thyroglobulin secretion.

Specific receptors for endothelin-1 (ET), a newly described vasoconstrictor peptide isolated from endothelium, have been identified in endocrine tissues such as hypothalamus, adrenal and pituitary. ET binding or action, not previously described in thyroid, were explored in this study. ET binding in cultured human thyrocytes was assayed at 4 C, 25 C, and 37 C, for 0.5-6 h with [125I]ET (0.1 nmol/L), and nonspecific binding estimated by coincubation with unlabeled ET (100 nmol/L). At 4 C, maximum specific binding was reached after 4 h; at 25 C and 37 C, specific binding increased in a time-dependent manner over 6 h with increased binding obtained at higher temperature. At 37 C after 2 h, 11% specific bound ET localized to surface membranes with 89% internalized. Scatchard analysis of surface membrane binding at 4 C for 4 h showed high affinity single class ET receptor (Kd = 0.20 nmol/L) and binding capacity of 4045 sites per cell. ET binding to thyroid cells had no effect on production of cAMP or cGMP. ET (0.1 nmol/L) significantly (P less than 0.001) inhibited thyroglobulin release from thyroid cells after 6 days with no effect on thymidine incorporation. Thus, we have identified specific receptors for endothelin in human thyrocytes, and an inhibitory action of the peptide on thyroglobulin release which is mediated by a noncyclic nucleotide mechanism.

Cells, Cultured↗
PubMed1992

Atrial natriuretic peptide receptors in human endometrial stromal cells.

Atrial natriuretic peptide (ANP) has been shown to affect water and ion transport and specific ANP binding has been identified in several secretory tissues. ANP commonly acts via stimulation of membrane-bound particulate guanylate cyclase with the production of cyclic guanosine monophosphate (cGMP). We questioned whether ANP played a role in the complex cyclic transformation of the endometrium into a secretory tissue, and whether its action was cGMP mediated. Endometrium was obtained by biopsy in regularly menstruating women and stromal cells were isolated and cultured for use in this study. ANP competitive binding assays were performed using 125I-labeled ANP (0.1 nmol/L) and increasing concentrations of unlabeled ANP (0-1000 nmol/L). Optimal binding was obtained after 3-h incubation at 4 C and binding characteristics, including dissociation constant and binding site quantity, were estimated by Scatchard analysis. Specific, high affinity (dissociation constant, 0.078 +/- 0.004 nmol/L) and low capacity (4,877 +/- 1,951 binding sites/cell) ANP binding was identified, with nonspecific binding representing less than or equal to 16% of total binding. Evaluation of ANP-stimulated cyclic nucleotide production revealed an increase in cGMP production, with a 7-fold increase at 1000 nmol/L ANP, and no effect on cAMP production. In conclusion, we have identified specific high affinity receptors for ANP in human endometrial cells, suggesting a role for ANP in endometrial cell function and/or development mediated via cGMP production. We propose that ANP may affect local salt and water metabolism, may be involved in the secretory evolution of glandular and stromal cells, and may further facilitate endometrial development via modulation of local vascular tone and endothelial permeability.

Atrial Natriuretic Factor↗
PubMed1991

Carotid body chemoreception in the absence and presence of CO2-HCO3-.

Carotid body (CB) chemosensory responses to natural and pharmacological stimuli were studied in vitro in the presence and nominal absence of CO2-HCO3- in the perfusion-superfusion media. The CBs obtained from cats (n = 10), anesthetized with sodium pentobarbitone, were simultaneously perfused and superfused with a modified Tyrode solution at 36.5 +/- 0.5 degrees C, equilibrated respectively with PO2 of 120 and less than 20 Torr. The Tyrode, nominally free of CO2-HCO3- (HEPES-NaOH, pH 7.38, 310 mOsm), was used first. Subsequently the Tyrode containing HEPES-HCO3-, equilibrated with PCO2 of 36.8 Torr (pH 7.38) was used. Chemosensory discharges were recorded from the carotid sinus nerve. Both hypoxia (PO2 = 20-25 Torr) and ischemic hypoxia stimulated the discharge in the absence and presence of CO2-HCO3-. However, the presence of CO2-HCO3- significantly raised the baseline activity, augmented the speed, sensitivity and the maximal responses to both types of hypoxia. Hypercapnic perfusate (PCO2 = 65 Torr at pH 7.17) produced a peak response equally promptly in the absence and presence of CO2-HCO3- in the ongoing perfusate but generated a larger and more sustained response. Presence of CO2-HCO3- strongly potentiated the responses to cyanide (10(-10)-10(-7) mol) but less strikingly the responses to nicotine (10(-11)-10(-8) mol). Thus, the extracellular CO2-HCO3- significantly improved the response to hypoxia but was not essential for O2 chemoreception. The underlying mechanisms of the effect of CO2-HCO3- is likely to be mediated by the Cl(-)-HCO3- anion exchanger in the pH regulation of glomus cells.

Animals↗
PubMed1991

Endogenous opiates and ventilatory acclimatization to chronic hypoxia in the cat.

The effects of the opiate antagonist naloxone (0.4 mg.kg-1, i.v.) on carotid chemoreceptor and ventilatory responses to graded steady-state levels of hypoxia and hypercapnia were investigated in two groups of cats: chronically normoxic and chronically hypoxic. The cats of the latter group were exposed to PIO2 of about 70 mm Hg at sea level for 3-4 weeks and showed an attenuated response to hypoxia. All cats were tested under alpha-chloralose anesthesia. Naloxone treatment did not increase appreciably carotid chemoreceptor activity or its responses to hypoxia and hypercapnia in either cat group. Naloxone caused a small ventilatory stimulation in the chronically hypoxic cats, so that the attenuated response to hypoxia was not relieved. By contrast, the chemoreflex ventilatory response to hypoxia was stimulated by naloxone in the chronically normoxic cats. The findings that the depressed ventilatory chemoreflexes in the chronically hypoxic cat were not ameliorated by the opiate antagonist indicate that an increased elaboration of endogenous opiates does not underlie ventilatory adaptation to chronic hypoxia.

Acclimatization↗
PubMed1991

Aortic and carotid body chemoreception in prolonged hyperoxia in the cat.

Carotid body chemosensory response to hypoxia is attenuated as a result of prolonged normobaric hyperoxia (NH) in the cat. The effect of NH is likely to be due to high cellular PO2 and O2-related free radicals. Accordingly, the effect would be less if O2 delivery to the chemoreceptor tissue could be compromised. The aortic bodies, which appear to have less of a circulatory O2 delivery, as suggested by their vigorous responses to a slight compromise of O2 flow compared with those of the carotid body, could provide a suitable testing material for the hypothesis. We tested the hypothesis by studying both aortic and carotid body chemoreceptors in the same cats (n = 6) which were exposed to nearly 100% O2 for about 60 h. These chemoreceptor organs were also studied in 6 control cats which were maintained in room air at sea-level. The cats were anesthetized and their carotid and aortic chemosensory fibers were identified by the usual procedure, and their responses to hypoxia and hypercapnia and to bolus injections (i.v.) of cyanide and nicotine were measured. In the NH cats, the carotid but not aortic chemosensory responses to hypoxia and cyanide were attenuated and to hypercapnia (both onset and steady state) augmented. The aortic chemoreceptors were stimulated by hypoxia, hypercapnia, cyanide and nicotine both in the NH and the control cats similarly. The results support the hypothesis that it is presumably a higher tissue blood flow and hence a higher concentration of O2-related free radicals which ultimately led to the specific attenuation of O2 chemoreception in the carotid body.

Analysis of Variance↗
PubMed1991

Carotid chemosensory timing effects on cervical sympathetic discharges in the cat.

The hypothesis that respiratory phase-related sympathetic nerve activity would manifest timing effect of carotid chemosensory input with respect to the central respiratory drive was tested in the anesthetized, paralyzed and artificially ventilated cats which were also vagotomized and tracheostomized. Preganglionic cervical sympathetic nerve fibers (PSNF) which were clearly responsive to carotid chemosensory stimulation were selected for the test. Simultaneously with the PSNF, phrenic nerve (PN) and internal intercostal expiratory nerve (IICEN) activities were also recorded. In most instances, carotid chemosensory discharges were monitored in order to register the precise timing of its input to the brain-stem. Timed stimulation of carotid chemoreceptors was elicited by bolus injections of cyanide (10-20 micrograms), nicotine (10-20 micrograms) and CO2-saturated saline (0.2-0.5 ml) into the base of the common carotid artery. Two types of PSNFs were identified: type I discharged only in synchrony with the PN activity (13/47) and type II fired independently of, but entrained to, PN activity (34/47). Type I displayed characteristic timing effects of carotid chemoreflex on PN discharges. However, the sympathetic activity did not share the after-discharge of PN which persisted beyond the carotid chemoreceptor stimulation. Type II did not manifest any timing effect of carotid chemoreflex with respect to PN and IICEN activities. These results suggest the following model of carotid chemoreflex effects on sympathetic neuron activities: a group is exclusively gated by the properties of central respiratory drive whereas the other is not gated but entrained by it in the spontaneously breathing anesthetized cat.

Anesthesia↗
PubMed1991

Sympathetic influence on carotid chemoreceptor response to substance P in the cat.

Previous studies have suggested that substance P (SP) may play a role in the carotid chemoreceptor response to hypoxia. Given the data from these studies we speculated that within the carotid body hypoxia might release SP which then acts on the chemosensitive unit. Concomitantly SP might be released in the superior cervical ganglion (SCG) and increase sympathetic outflow to the carotid body by interacting with acetylcholine in the SCG. The resulting vasoconstriction in the carotid body would further increase neural output from the carotid body. Hence we hypothesized that the exogenous SP on the carotid chemoreceptor neural activity would decrease after eliminating preganglionic inflow into the SCG. The hypothesis was tested using anesthetized, paralyzed and artificially ventilated cats. Neural activity from the carotid body (carotid chemoreceptor activity) or from the SCG (ganglioglomerular efferent nerve activity (GGN)) was measured. Close intra-arterial administration of SP (10 micrograms) caused a sustained stimulation of the carotid chemoreceptor activity which was accompanied by a fall in arterial blood pressure. The magnitude and time-course of the carotid body responses were extremely variable among the cats. The duration of increased chemoreceptor activity was significantly shortened after a transection of the cervical sympathetic nerve (CVSN). As a control, the duration of carotid body stimulation produced by the second injection of SP in a group of sham-operated cats was measured. This was essentially the same as the first injection, suggesting that the tachyphylactic effect of SP was negligible. The effects of the commonly used pharmacological agents (nicotine, cyanide, dopamine) on carotid chemoreceptor activity were not affected by the transection of the CVSN, GGN activity was also increased by exogenous SP. These results suggest that the effect of exogenous SP on carotid chemoreceptor activity consists of two components: (1) an initial direct excitatory effect; (2) a slowly developing excitatory effect mediated by the sympathetic outflow to the carotid body. The effects could be augmented by the accompanying hypotension.

Animals↗
PubMed1991

Chronic cobalt causes hypertrophy of glomus cells in the rat carotid body.

We tested the hypothesis that chronic cobalt administration would induce carotid body cellular response along with polycythemia as found in chronic hypoxia if common oxygen-sensitive mechanisms were involved in the two instances. Morphometric studies were performed on carotid bodies in male rats that were chronically treated with cobalt chloride (0.17 mumol/kg, ip, daily for 6 wk) and in control rats that received blank saline injections. The rats were anesthetized, blood samples were collected for hematocrit, and the carotid bodies were surgically exposed and were perfused and superfused with the buffered fixative (3% glutaraldehyde plus 1% paraformaldehyde, pH 7.40, 330-340 mosM). The carotid bodies were processed, and ultrathin sections were cut for electron microscopy and morphometry of type I (glomus) and type II cells. Hematocrit increased from 44% in the control to 74% in the cobalt-treated rats, and the mean volume of type I cells increased from 424 to 1,061 microns 3. Type II cells did not show any significant change in size. The results suggest that cobalt stimulated oxygen-sensitive mechanism in the glomus cells of the carotid body and that the glomus cell is a site of oxygen chemosensing.

Animals↗
PubMed1991

Carbonic anhydrase and chemoreception in the cat carotid body.

To test the hypothesis that CO2 and O2 chemoreception in the carotid body (CB) may depend on its carbonic anhydrase (CA) activity, we used an in vitro cat CB preparation and studied the effects of methazolamide, a permeable CA inhibitor (pK 7.3), on the chemosensory responses to CO2, O2, and nicotine. The isolated CB was perfused and superfused with Tyrode solution, free of CO2-HCO3-, at 36.0 +/- 0.5 degrees C. The frequency of chemosensory discharges was recorded from the whole carotid sinus nerve. The responses to bolus injections (0.3-0.5 ml) of Tyrode solution equilibrated with PCO2 of 38-110 Torr, switching from HEPES to CO2-HCO3- Tyrode (PCO2 = 25-60 Torr) for about 3 min, hypoxic Tyrode (PO2 = 25-30 Torr) for 2-8 min, perfusate flow interruptions for approximately 4 min, and bolus injections of nicotine (4 nmol) were studied before, during, and after perfusion (30-45 min) with methazolamide (42.4 microM). Methazolamide reversibly inhibited, delayed, and reduced the responses to transient CO2 stimulus, diminished the onset of but not the late response to prolonged CO2 stimulus, and delayed but did not decrease the responses to hypoxia and perfusate interruption. The response to nicotine did not change. The results indicated that CA in the glomus cells played a crucial role primarily in the speed and magnitude of the initial response to CO2 stimulus and indirectly influenced O2 chemoreception. These effects were upstream from the nicotine receptor-mediated sensory response.

Animals↗
PubMed1991

Optical measurements of the dependence of chemoreception on oxygen pressure in the cat carotid body.

The relationship between oxygen pressure (PO2) in the carotid body and carotid sinus nerve discharge was evaluated in the isolated perfused/superfused cat carotid body using the oxygen-dependent quenching of phosphorescence. Images of phosphorescence intensity arising from Pd-coproporphyrin within the microcirculation of the carotid body provided measurements of intravascular PO2. These measurements were substantiated by determining phosphorescence life-time. The carotid body was perfused in the isolated state via the common carotid artery with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Tyrode solution, pH 7.4, at a constant pressure of 80 mmHg. Superfusion was maintained with similar media equilibrated with 100% argon. PO2 in the exchange vessels was markedly less than that in the perfusate entering the carotid artery, 23 +/- 3 and 45 +/- 3 Torr for normoxic (111 +/- 15 Torr) and hyperoxic (345 +/- 72 Torr) perfusates, respectively. Chemosensory discharge rose slowly in response to a brief interruption of perfusate flow as PO2 steadily declined from either of these capillary PO2 values to approximately 10 Torr. Between approximately 10 and 3 Torr, chemosensory discharge increased strikingly, concomitant with an enhanced rate of oxygen disappearance, from -36 +/- 4 to -69 +/- 13 (92% change) and -28 +/- 3 to -48 +/- 3 (71% change) Torr/s for normoxic and hyperoxic perfusates, respectively. As PO2 fell below approximately 3 Torr, oxygen disappearance slowed and neural activity decayed. Thus the relationships between microvascular PO2 and chemosensory discharge and between oxygen disappearance and neural discharge suggest that oxygen metabolism in the carotid body determines the expression of oxygen chemoreception.

Animals↗
PubMed1991

In vitro perfused-superfused cat carotid body for physiological and pharmacological studies.

An in vitro perfused carotid body preparation was developed to study its chemosensory responses to physiological and pharmacological stimuli. The carotid bifurcation with the carotid body was vascularly isolated and excised from pentobarbital sodium-anesthetized cats. The CB was perfused in a chamber by gravity (80 Torr) with modified Tyrode's solution (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-NaOH at pH 7.40) equilibrated at a given Po2 and superfused with the same medium at (Po2 of 20 Torr). The temperature was maintained at 35.5 +/- 0.5 degrees C. The frequency of chemosensory discharges (CD) was recorded from the whole carotid sinus nerve (n = 24), and the responses were tested by repeated interruptions of perfusate flow (SF), perfusion with hypoxic medium, and injections of nicotine and cyanide (0.1 nmol to 1 mumol) and hypercapnic medium. During hyperoxic perfusion, SF resulted in a sigmoidal increase in CD, reaching a maximum that was 23.6 +/- 4.4-fold greater than the basal activity. Restoration of flow returned CD promptly to basal values. After normoxic perfusion, SF led to a similar maximal activity more rapidly, but the duration was shorter. Reduction of the perfusate PO2 (Po2 from 450 Torr to 150, 30, and less than 10 Torr) caused a nonlinear increase in CD. CO2 stimuli (PCo2 38-110 Torr) resulted in a linear increase in CD. Nicotine or cyanide increased CD in a dose-dependent manner. The preparation retained its initial responsiveness for 2-3 h, making extensive experimental studies feasible.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Thyrotropin modulates receptor-mediated processing of the atrial natriuretic peptide receptor in cultured thyroid cells.

In a prior study of atrial natriuretic peptide (ANP) binding to cultured thyroid cells, we reported that at 4 C, more than 95% of bound ANP is recovered on cell membranes, with negligible ANP internalization observed. Since ANP binding was inhibited by TSH, we have further studied TSH effects on postbinding ANP processing to determine whether this phenomenon reflects enhanced endocytosis of the ANP-receptor complex. An ANP chase study was initiated by binding [125I] ANP to thyroid cells at 4 C for 2 h, followed by incubation at 37 C. ANP processing was then traced by following 125I activity at various time intervals in three fractions: cell surface membranes, incubation medium, and inside the cells. Radioactivity released into medium represented processed ANP rather than ANP dissociated from surface membranes, since prebound [125I]ANP could not be competitively dissociated by a high concentration of ANP (1 mumol/L) at 37 C. Chase study results showed that prebound ANP quickly disappeared from cell membranes down to 34% by 30 min. Internalized ANP peaked at 10 min, with 21% of initial prebound ANP found inside the cells. At the same time, radioactivity recovered in incubation medium sharply increased between 10-30 min from 8% to 52%. Preincubation of cells with chloroquine (which blocks degradation of the ANP-receptor complex by inhibiting lysosomal hydrolase) caused a 146% increase in internalized [125I]ANP by 30 min (39% compared to 15% control), while medium radioactivity decreased from 52% to 16%, suggesting that processing of the receptor complex is mediated via lysosomal enzymes. In chase studies employing cells pretreated with chloroquine, TSH stimulated the internalization rate of ANP-receptor complex. By 30 min, TSH significantly reduced the membrane-bound ANP, and the decrease was inversely correlated to the increase in internalized radioactivity. In the absence of ANP ligand, TSH also modulated receptor translocation, resulting in decreased receptor number on surface membrane. However, TSH did not affect ANP degradation on the basis of analysis by immunoprecipitation and high performance liquid chromatography. Taken together, these data suggest that TSH downregulates ANP receptor on thyroid cell surface membranes, and that an ANP degradative pathway exists which is not mediated through receptor binding.

Cells, Cultured↗