Inhibition of PMA-induced, LFA-1-dependent lymphocyte aggregation by ADP ribosylation of the small molecular weight GTP binding protein, rho.

Botulinum C3 exoenzyme specifically ADP-ribosylates a group of ras-related small molecular weight GTP-binding proteins, rho, and inhibits their biological activity. Using this enzyme, we examined the function of rho in PMA-induced activation of lymphocyte function-associated antigen-1 (LFA-1) in a B lymphoblastoid cell line, JY. Northern blot analysis revealed that among the three rho genes, rhoA mRNA was predominantly expressed in JY cells. Consistently, only one [32P]ADP-ribosylated band was found when the lysate of the cells was subjected to ADP ribosylation by C3 exoenzyme. When the cells were cultured with C3 exoenzyme, this substrate was ADP-ribosylated in situ in a time- and concentration-dependent manner. Concomitant with this ADP ribosylation, PMA-induced LFA-1/intercellular adhesion molecule (ICAM)-1-dependent aggregation of JY cells was inhibited. This inhibition was blocked by prior treatment of the enzyme with an anti-C3 monoclonal antibody, and overcome by stimulation with higher concentrations of PMA. The C3 exoenzyme-induced inhibition was not affected by shaking of the cell suspension, while inhibition of aggregation by cytochalasin B was abolished by this procedure, suggesting that the inhibitory effect of the C3 exoenzyme treatment was not due to decrease in cell motility. The C3 exoenzyme treatment affected neither protein phosphorylation in JY cells before and after PMA stimulation, nor affected surface expression of LFA-1 and ICAM-1. These results suggest that rhoA protein works downstream of protein kinase C activation linking PMA stimulation to LFA-1 activation and aggregation in JY cells.

Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho.

Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA- and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPA-induced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.

Protein kinase N (PKN) and PKN-related protein rhophilin as targets of small GTPase Rho.

The Rho guanosine 5'-triphosphatase (GTPase) cycles between the active guanosine triphosphate (GTP)-bound form and the inactive guanosine diphosphate-bound form and regulates cell adhesion and cytokinesis, but how it exerts these actions is unknown. The yeast two-hybrid system was used to clone a complementary DNA for a protein (designated Rhophilin) that specifically bound to GTP-Rho. The Rho-binding domain of this protein has 40 percent identity with a putative regulatory domain of a protein kinase, PKN. PKN itself bound to GTP-Rho and was activated by this binding both in vitro and in vivo. This study indicates that a serine-threonine protein kinase is a Rho effector and presents an amino acid sequence motif for binding to GTP-Rho that may be shared by a family of Rho target proteins.

Synthesis of atrial natriuretic polypeptide in human failing hearts. Evidence for altered processing of atrial natriuretic polypeptide precursor and augmented synthesis of beta-human ANP.

To elucidate the synthesis of atrial natriuretic polypeptide (ANP) in the failing heart, 20 human right auricles obtained at cardiovascular surgery were studied. The concentration of alpha-human ANP-like immunoreactivity (alpha-hANP-LI) in human right auricles ranged from 13.8 to 593.5 micrograms/g, and the tissue alpha-hANP-LI concentration in severe congestive heart failure (CHF) (New York Heart Association [NYHA] functional class III and class IV) (235.4 +/- 57.2 micrograms/g) was much higher than that in mild CHF (NYHA class I and class II) (52.5 +/- 15.6 micrograms/g). Atrial alpha-hANP-LI levels were significantly correlated with plasma concentrations of alpha-hANP-LI in these patients (r = 0.84, P less than 0.01). High performance gel permeation chromatography and reverse phase high performance liquid chromatography coupled with radioimmunoassay for ANP revealed that the alpha-hANP-LI in the human auricle consisted of three major components of ANP, gamma-human ANP (gamma-hANP), beta-human ANP (beta-hANP) and alpha-human ANP (alpha-hANP). Comparing percentages of gamma-hANP, beta-hANP, and alpha-hANP in alpha-hANP-LI in severe CHF with those in mild CHF, the predominant component of alpha-hANP-LI was gamma-hANP in mild CHF, whereas beta-hANP and/or alpha-hANP were prevailing in severe CHF and, especially, beta-hANP was markedly increased in human failing hearts. These results demonstrate that the total ANP concentration in the atrium of the human heart is increased in severe CHF and that the increase of ANP in the human failing heart is mainly due to the increase of small molecular weight forms of ANP, beta-hANP, and alpha-hANP, especially beta-hANP, and indicate that the processing of ANP precursor, or gamma-hANP, in the human failing heart differs from that in the normal heart, suggesting that the failing heart augments synthesis and secretion of ANP as one of its own compensatory responses.

Chronic blockade of endogenous atrial natriuretic polypeptide (ANP) by monoclonal antibody against ANP accelerates the development of hypertension in spontaneously hypertensive and deoxycorticosterone acetate-salt-hypertensive rats.

To explain the pathophysiological significance of endogenous atrial natriuretic polypeptide (ANP) in the development of hypertension, we examined the effect of chronic, repetitive administrations of MAb raised against alpha-rat ANP in two rat models of hypertension, spontaneously hypertensive rats of the stroke prone substrain (SHR-SP), and deoxycorticosterone acetate (DOCA)-salt rats. Weekly intravenous administrations of MAb with high affinity for alpha-rat ANP, named KY-ANP-II (MAb[KY-ANP-II]), started at the age of 6 wk, significantly augmented the rise in blood pressure of SHR-SP, compared with control SHR-SP treated with another MAb with quite low affinity for alpha-rat ANP, named KY-ANP-I (MAb[KY-ANP-I]), throughout the observation period. The administrations of MAb[KY-ANP-II] had no significant effect on blood pressure of age-matched normotensive Wistar Kyoto rats, compared with those receiving MAb[KY-ANP-I]. Weekly administrations of MAb[KY-ANP-II] also significantly aggravated hypertension in DOCA-salt rats. Blood pressure of DOCA-salt rats treated with MAb[KY-ANP-II] was significantly higher than that of DOCA-salt rats treated with MAb[KY-ANP-I] throughout 8 wk of DOCA and 1% saline administration. The administration of MAb[KY-ANP-II] also significantly attenuated exaggerated diuresis and natriuresis in DOCA-salt rats compared with those treated with MAb[KY-ANP-I]. Elevated plasma cGMP levels of both SHR-SP and DOCA-salt rats were significantly reduced by the administration of MAb[KY-ANP-II]. These results suggest the compensatory role of augmented secretion of ANP in these hypertensive rats and support the concept that augmented secretion of ANP could represent an antihypertensive deterrent mechanism.

Activation of the osmo-sensitive chloride conductance involves P21rho and is accompanied by a transient reorganization of the F-actin cytoskeleton.

Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume. Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles. In addition, an increase in total cellular F-actin was observed. Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho. In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity. Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux. Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.

ADP-ribosylation of the rhoA gene product by botulinum C3 exoenzyme causes Swiss 3T3 cells to accumulate in the G1 phase of the cell cycle.

Using botulinum C3 exoenzyme, which specifically ADP-ribosylates the rho gene products (rho proteins), we examined the role of these proteins in cell cycle progression in Swiss 3T3 cells. Incubation of cell lysates with C3 exoenzyme revealed a single [32P]ADP-ribosylated protein with an M(r) of 23K. This protein was identified as rhoA protein by isoelectric focusing and peptide mapping. When C3 exoenzyme was added to the culture, it ADP-ribosylated the substrate protein in the cells and reduced their growth rate and saturation density. The reduction was dependent on the amount of C3 exoenzyme and on the extent of ADP-ribosylation of the rho protein in the cells. Flow cytometric analysis of logarithmically growing cells showed that the enzyme treatment concentration-dependently accumulated the cells in the G1 phase of the cell cycle. When G1-enriched cells were treated with C3 exoenzyme and cell cycle progression initiated by the addition of serum was monitored, inhibition of G1-S transition was clearly observed. These results suggest that the rhoA gene product plays a critical role in G1-S progression in cultured Swiss 3T3 cells and that the ADP-ribosylation abolishes this activity and causes the cells to accumulate in G1 phase.

The involvement of the rho gene product, a small molecular weight GTP-binding protein, in polyploidization of a human megakaryocytic cell line, CMK.

The role of rho proteins, which are ras p21-related small GTP-binding proteins, in megakaryocyte endomitosis was examined using a botulinum C3 exoenzyme (C3), a rho inactivating enzyme. The megakaryocytic leukemia cell line CMK expressed high levels of rhoA and rhoC mRNAs, whereas rhoB mRNA was expressed at a very low level. The addition of C3 to the culture medium caused ADP-ribosylation of the rho proteins in CMK cells in a dose- and time-dependent manner. This procedure also induced a higher frequency of polyploid cells with increased glycoprotein (GP) IIb/IIIa antigens on the cells. This effect of C3 on both ploidy and the antigen expression was abolished by prior incubation of C3 with an anti-C3 monoclonal antibody. Cytochalasin B, an actin polymerization inhibitor, also induced polyploid cells; however, it did not stimulate the expression of GP IIb/IIIa antigens in CMK cells. This finding suggests that C3-induced increase in the expression of GP IIb/IIIa antigens was not through the actin microfilament disassembly. The present study suggests that the rho p21 is a partly regulatory component in polyploidization and GP IIb/IIIa antigen expression of a human megakaryocytic cell line, CMK.

Inhibitory action of leumorphin on vasopressin secretion in conscious rats.

To elucidate the role of leumorphin, a kappa-agonist derived from proenkephalin-B (neoendorphin/dynorphin precursor), in the control of arginine vasopressin (AVP) secretion, we examined the effects of intracerebroventricular (icv) administration of leumorphin on AVP secretion under basal and stimulated conditions in conscious unrestrained rats. Intracerebroventricular injection of leumorphin (60 or 600 pmol) significantly inhibited basal AVP secretion. In 72-h water-deprived rats, icv injection of leumorphin (60 or 600 pmol) also suppressed AVP secretion in a dose-dependent manner. The AVP response induced by icv injection of angiotensin II (100 pmol) was significantly decreased by the simultaneous icv injection of leumorphin (6-600 pmol) in a dose-dependent manner. Intracerebroventricular administration of leumorphin (600 pmol) also reduced the AVP secretion stimulated by icv injection of carbachol (50 pmol). Intravenous pretreatment with naloxone (0.5 mg/kg BW) diminished the inhibitory action of leumorphin (60 pmol) on AVP secretion. However, no effect on AVP secretion was observed after iv injection of leumorphin (600 pmol). These results indicate that leumorphin possesses a potent inhibitory effect on AVP secretion, suggesting its important role in the regulation of AVP secretion in the brain.

Modulatory role of vasopressin in secretion of atrial natriuretic polypeptide in conscious rats.

To investigate whether vasopressin is involved in the secretory mechanism of atrial natriuretic polypeptide (ANP), effects of arginine-vasopressin (AVP) administered iv on plasma ANP levels were studied in conscious, unrestrained rats. The administration of 100 ng and 1 microgram of AVP caused a dose-dependent increase of the plasma ANP level, which was blocked by a V1-receptor antagonist of AVP, and was attenuated by 5 ml blood volume reduction before the stimulation. The injection of less than 10 ng of AVP induced no significant effects on ANP secretion. However, the administration of 5 ng of AVP significantly enhanced ANP secretion induced by intravascular volume expansion with 3 ml saline infusion. These results suggest the possible physiological significance of AVP as a modulator rather than a direct stimulator of ANP secretion from the heart.

A monoclonal antibody to alpha-human atrial natriuretic polypeptide.

A monoclonal antibody to alpha-human atrial natriuretic polypeptide (alpha-hANP), KY-ANP-I, has been produced by fusion of a nonproducing mouse myeloma cell line, X63-Ag8.653, with spleen cells from BALB/c mice immunized with synthetic alpha-hANP conjugated to bovine thyroglobulin using the carbodiimide coupling procedure. Hybridomas were screened for antibody production by radioimmunoassay using culture media and 125I-alpha-hANP. They were cloned by the limiting dilution technique, expanded in culture, and injected intraperitoneally into BALB/c mice. The obtained antibody belonged to the immunoglobulin G1 subclass. Analysis by a Scatchard plot revealed a high affinity for alpha-hANP, with an association constant of 3.1 x 10(10) M-1. With this monoclonal antibody, a specific radioimmunoassay for alpha-hANP has been established. The antibody in mouse ascites was available for radioimmunoassay at a final dilution of 1:10(6). Values of IC10 and IC50 in this radioimmunoassay were 3 and 30 fmol/tube, respectively. The radioimmunoassay showed a cross-reactivity of 0.9% with alpha-rat ANP. alpha-hANP-(8-22) and alpha-ANP-(1-6) exhibited less cross-reactivity than alpha-rat ANP on a molar basis. There was no cross-reaction with alpha-ANP-(17-28). Thus, the recognized epitope must be located in the N-terminal half of the ring structure of alpha-hANP including Met12 residue. This radioimmunoassay could detect gamma-hANP and beta-hANP as well as alpha-hANP. The monoclonal antibody was also useful for immunohistochemical studies. ANP-positive cells were finely stained in the human atrium using the avidin-biotin-peroxidase complex technique.(ABSTRACT TRUNCATED AT 250 WORDS)

Implication of leumorphin in inhibitory control of vasopressin secretion in conscious rats.

The effects of leumorphin, a kappa-agonist derived from proenkephalin B (neoendorphin and dynorphin precursor), on vasopressin secretion were studied under basal and stimulated conditions in conscious, unrestrained rats. Intracerebroventricular injection of leumorphin (60 or 600 pmol) significantly inhibited basal vasopressin secretion. The vasopressin response induced by intracerebroventricular injection of angiotensin II (100 pmol) was significantly suppressed, in a dose-dependent fashion, by the simultaneous intracerebroventricular injection of leumorphin (6, 60, or 600 pmol). Intravenous pretreatment with naloxone (0.5 mg/kg body weight) diminished the inhibitory action of leumorphin (60 pmol) on vasopressin secretion. Moreover, naloxone (0.5 mg/kg body weight) prolonged the vasopressin secretion induced by intracerebroventricular injection of angiotensin II (100 pmol). These results indicate that leumorphin possesses a potent inhibitory effect on vasopressin secretion and that, alone or in combination with other endogenous opioid peptides, it plays an important role in the control of vasopressin secretion.

Atrial natriuretic factor in essential hypertension and adrenal disorders.

Patients with untreated essential hypertension had significantly higher plasma atrial natriuretic factor (ANF) levels (92.9 +/- 12.9 pg/ml, mean +/- SE) than those of age-matched controls (37.8 +/- 6.0 pg/ml; p less than 0.01). Plasma ANF levels in essential hypertensive patients showed a significant positive correlation with mean arterial pressure (MAP; r = 0.46, p less than 0.05) and an inverse correlation with plasma renin activity (PRA; r = -0.43, p less than 0.05). Plasma ANF levels after medication showed significant correlation with the decrease in MAP (r = 0.565, p less than 0.05). Patients with primary aldosteronism had significantly higher plasma ANF levels (122.4 +/- 30.2 pg/ml, n = 8) than those of controls (p less than 0.05). The levels returned to normal after extirpation of adrenal tumors. The response of plasma ANF levels in patients with primary aldosteronism to volume expansion with infusion of 2 L of physiological saline in 2 hours was greater than in controls. Such exaggerated response disappeared after surgical treatment. Infusion of angiotensin II (Ang II; 20 ng/kg/min) or norepinephrine (200 ng/kg/min) for 30 minutes to normal volunteers (n = 5) resulted in a rise in MAP (24.9 +/- 3.3 and 15.8 +/- 4.4 mm Hg, respectively) and a twofold increase in plasma ANF level. Infusion of the Ang II antagonist [Sar1, Ile8]Ang II (600 ng/kg/min) for 30 minutes, resulted in a rise in MAP (18.8 +/- 2.1 mm Hg) and more than a twofold increase in plasma ANF level in patients with essential hypertension (n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of N-terminal fragment of gamma-human atrial natriuretic polypeptide.

To elucidate the posttranslational processing of gamma-human atrial natriuretic polypeptide (human atrial natriuretic factor-[1-126]), which is a prohormone of alpha-human atrial natriuretic polypeptide (human atrial natriuretic factor-[99-126]), and the secretion of gamma-human atrial natriuretic polypeptide-derived peptides from the heart, we established a radioimmunoassay specific for the N-terminal sequence of gamma-human atrial natriuretic polypeptide, gamma-human atrial natriuretic polypeptide-(1-25), as well as a radioimmunoassay for alpha-human atrial natriuretic polypeptide. With the aid of the radioimmunoassays for gamma-human atrial natriuretic polypeptide-(1-25) and for alpha-human atrial natriuretic polypeptide, we detected 290 +/- 35.6 pg/ml of gamma-human atrial natriuretic polypeptide-(1-25)-like immunoreactivity in plasma from healthy humans, while the simultaneously determined plasma alpha-human atrial natriuretic polypeptide-like immunoreactivity level was 20.9 +/- 2.8 pg/ml. Correlation between the two values was significant. High performance gel permeation chromatographic analysis revealed that the plasma gamma-human atrial natriuretic polypeptide-(1-25)-like immunoreactivity was composed of a component (molecular weight, 10,000) without alpha-human atrial natriuretic polypeptide-like immunoreactivity, while the plasma alpha-human atrial natriuretic polypeptide-like immunoreactivity was composed of alpha-human atrial natriuretic polypeptide with a molecular weight of 3000. In patients with heart diseases, the plasma gamma-human atrial natriuretic polypeptide-(1-25)-like immunoreactivity level showed a concomitant and graded increase, with the plasma alpha-human atrial natriuretic polypeptide-like immunoreactivity level in agreement with the severity of the disease. There were significant positive correlations between the two immunoreactivity levels and right or left atrial pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain renin-angiotensin. Central control of secretion of atrial natriuretic factor from the heart.

To elucidate the modulatory role of the brain renin-angiotensin system in the regulation of the secretion of atrial natriuretic factor (ANF) from the heart, the effects of intracerebroventricular administration of angiotensin II on the plasma ANF level were examined in conscious unrestrained rats. Administration of angiotensin II in doses of 100 ng and 1 microgram significantly enhanced ANF secretion induced by volume loading with infusion of 3 ml of saline (peak values of the plasma ANF level: control, 220 +/- 57 pg/ml; angiotensin II 100 ng, 1110 +/- 320 pg/ml, p less than 0.01; angiotensin II 1 microgram, 1055 +/- 60 pg/ml, p less than 0.01). Injection of angiotensin II alone had no significant effect on the basal plasma ANF level. Central angiotensin II-induced ANF secretion was significantly attenuated by pretreatment with intravenous administration of the V1-receptor antagonist of vasopressin or intracerebroventricular administration of phentolamine. These results indicate that the brain renin-angiotensin system modulates ANF secretion in response to volume loading through the stimulation of vasopressin secretion or the activation of the central alpha-adrenergic neural pathway.

Atrial natriuretic polypeptide in bovine adrenal medulla.

Two radioimmunoassays for alpha-human atrial natriuretic polypeptide (alpha-hANP) with different specificities were used to study the tissue level and the nature of alpha-hANP-like immunoreactivity in the bovine adrenal gland. A considerable amount of alpha-hANP-like immunoreactivity was detected in the adrenal medulla (90.8 +/- 21.1 and 90.0 +/- 23.1 ng/g with the two radioimmunoassays), while no detectable amount (less than 1.0 ng/g) was present in the cortex. Gel permeation chromatographic analysis showed that ANP in the medulla is composed of two components of alpha-hANP-like immunoreactivity with high and low molecular weights in the approximate ratio of 2:1, eluting at the elution positions of gamma-hANP and alpha-hANP, respectively. Reverse-phase high performance liquid chromatographic analysis revealed that alpha-hANP-like immunoreactivity with a low molecular weight in the medulla consists of two major components, which comigrate with synthetic alpha-hANP(5-28) and alpha-hANP. When cultured bovine adrenal chromaffin cells were incubated in the presence of nicotine (10(-5) M), alpha-hANP-like immunoreactivity was released into the medium concomitantly with catecholamines from chromaffin cells. These findings indicate that a discrete ANP system is present in the adrenal medulla and that ANP is cosecreted with catecholamines from chromaffin cells, suggesting the possible involvement of ANP in the adrenomedullary function.

Effects of intravenously administered beta-human atrial natriuretic polypeptide in humans.

beta-Human atrial natriuretic polypeptide (beta-hANP) is an antiparallel dimer of alpha-human ANP (alpha-hANP) that was isolated from human atria. Using synthetic beta-hANP and a radioimmunoassay for alpha-hANP that also detects beta-hANP, we have previously demonstrated that beta-hANP is converted into alpha-hANP in human plasma in vitro. In the present study, we compared the effects of intravenous administration of beta-hANP (100 micrograms) to five normal human volunteers with those of an equimolar administration of alpha-hANP (50 micrograms) to the same subjects, and we also investigated the possible mechanisms of actions of beta-hANP. Although the administration of alpha-hANP caused a significant decrease in blood pressure with a reactional increase of heart rate, beta-hANP elicited minimal change of blood pressure. In contrast, beta-hANP exerted more potent and longer lasting diuretic and natriuretic activities than did alpha-hANP. Net changes in urine volume and sodium excretion induced by beta-hANP (579 +/- 65 ml, 56.0 +/- 9.9 mEq) were significantly greater than those elicited by alpha-hANP (396 +/- 50 ml, 34.7 +/- 4.9 mEq; p less than 0.05, respectively). The administration of beta-hANP revealed a longer retention of the ANP-like immunoreactivity level in plasma, compared with that of alpha-hANP. High performance gel permeation chromatography coupled with the radioimmunoassay revealed that beta-hANP (Mr = 6000) was also converted into alpha-hANP (Mr = 3000) in human plasma in vivo. The demonstrated conversion of beta-hANP into alpha-hANP could be relevant to the observed effects of beta-hANP in humans.

Gamma-atrial natriuretic polypeptide (gamma ANP)-derived peptides in human plasma: cosecretion of N-terminal gamma ANP fragment and alpha ANP.

Using RIAs for the N- and C-terminal fragments of the human atrial natriuretic polypeptide (ANP) precursor gamma ANP, that is gamma ANP-(1-25), and alpha ANP [gamma ANP-(99-126)], we studied the secretion of gamma ANP-derived peptides from the heart in normal subjects and patients with heart disease, chronic renal failure, and cirrhosis. We detected gamma ANP-(1-25)-like immunoreactivity (-LI) in plasma from normal subjects (n = 17) in considerable amounts [mean, 510 +/- 62 (+/- SE) pg/mL (174 +/- 21 pmol/L)]; the mean plasma alpha ANP-LI level at the same time in these subjects was 32.8 +/- 4.4 pg/mL (10.7 +/- 1.4 pmol/L). Gel permeation chromatographic analysis of plasma samples from normal subjects and patients with heart disease and chronic renal failure revealed two major components; one was alpha ANP, and the other was the 10K N-terminal gamma ANP fragment (N-peptide) resulting from the removal of alpha ANP (3K) from gamma ANP (13K). In addition, gamma ANP (13K), which possessed both gamma ANP-(1-25)-LI and alpha ANP-LI, and beta ANP, an antiparallel dimer of alpha ANP, were detected in some patients as minor components. A significant positive correlation between plasma levels of the N-terminal gamma ANP fragment and alpha ANP (P less than 0.01) and almost equal step-ups in the coronary sinus plasma levels of the N-terminal gamma ANP fragment and alpha ANP suggest that they are cosecreted in equimolar amounts. The high molar ratio of plasma gamma ANP-(1-25)-LI to alpha ANP-LI (17.4 +/- 1.4) in normal subjects and the significantly higher ratio in patients with chronic renal failure (36.9 +/- 7.1; P less than 0.01) suggest the slower clearance of the N-terminal gamma ANP fragment than alpha ANP and a role for the kidney in its degradation. Since the molar ratio of plasma gamma ANP-(1-25)-LI to alpha ANP-LI in patients with cirrhosis (20.7 +/- 2.7) was similar to that in normal subjects, it is unlikely that the N-terminal gamma ANP fragment is metabolized by the liver. In patients with heart disease, plasma gamma ANP-(1-25)-LI and alpha ANP-LI levels were higher in those with cardiac decompensation and were positively correlated with right atrial pressure, pulmonary arterial pressure, and pulmonary capillary wedge pressure, indicating cosecretion of the N-terminal gamma ANP fragment and alpha ANP in response to atrial stretch.(ABSTRACT TRUNCATED AT 400 WORDS)

Botulinum C3 exoenzyme blocks the tyrosine phosphorylation of p125FAK and paxillin induced by bombesin and endothelin.

In this study we examined the role of rho p21 in neuropeptide-stimulated tyrosine phosphorylation. Intact Swiss 3T3 cells were treated with the Clostridium botulinum C3 exoenzyme which specifically ADP ribosylates and inactivates rho p21. C3 exoenzyme treatment of cells caused a marked decrease in both bombesin- and endothelin-stimulated tyrosine phosphorylation of multiple proteins, including p125 focal adhesion kinase (FAK) and paxillin. Our results suggest that rho p21 is a component of the signal transduction pathway linking seven transmembrane domain receptors with tyrosine phosphorylation and cytoskeletal events.

Lysophosphatidic acid induces tyrosine phosphorylation and activation of MAP-kinase and focal adhesion kinase in cultured Swiss 3T3 cells.

Lysophosphatidic acid (LPA) added to serum-starved Swiss 3T3 cells induced, in a time- and concentration-dependent manner, tyrosine phosphorylation of multiple proteins, including proteins of 43, 64, 88 kDa and a group of proteins between 110 and 130 kDa. Among them, two proteins, p43 and p120, were identified as mitogen-activated protein kinase (MAP-kinase) and focal adhesion kinase (FAK), respectively, by immunoprecipitation and immunoblot analysis. Tyrosine phosphorylation of p64 peaked at 1 min and declined rapidly, whereas that of MAP-kinase and FAK peaked at 5 and 10 min after the addition of LPA, respectively. The activity of MAP-kinase determined as phosphorylation of myelin basic protein increased transiently about 3-fold at 5 min, and correlated with tyrosine phosphorylation. These results indicate that tyrosine phosphorylation of these proteins is a part of the signal transduction by LPA and may be involved in its mitogenic responses.