MAP2 is differentially phosphorylated in schizophrenia, altering its function.

Schizophrenia (Sz) is a highly polygenic disorder, with common, rare, and structural variants each contributing only a small fraction of overall disease risk. Thus, there is a need to identify downstream points of convergence that can be targeted with therapeutics. Reduction of microtubule-associated protein 2 (MAP2) immunoreactivity (MAP2-IR) is present in individuals with Sz, despite no change in MAP2 protein levels. MAP2 is phosphorylated downstream of multiple receptors and kinases identified as Sz risk genes, altering its immunoreactivity and function. Using an unbiased phosphoproteomics approach, we quantified 18 MAP2 phosphopeptides, 9 of which were significantly altered in Sz subjects. Network analysis grouped MAP2 phosphopeptides into three modules, each with a distinct relationship to dendritic spine loss, synaptic protein levels, and clinical function in Sz subjects. We then investigated the most hyperphosphorylated site in Sz, phosphoserine1782 (pS1782). Computational modeling predicted phosphorylation of S1782 reduces binding of MAP2 to microtubules, which was confirmed experimentally. We generated a transgenic mouse containing a phosphomimetic mutation at S1782 (S1782E) and found reductions in basilar dendritic length and complexity along with reduced spine density. Because only a limited number of MAP2 interacting proteins have been previously identified, we combined co-immunoprecipitation with mass spectrometry to characterize the MAP2 interactome in mouse brain. The MAP2 interactome was enriched for proteins involved in protein translation. These associations were shown to be functional as overexpression of wild type and phosphomimetic MAP2 reduced protein synthesis in vitro. Finally, we found that Sz subjects with low MAP2-IR had reductions in the levels of synaptic proteins relative to nonpsychiatric control (NPC) subjects and to Sz subjects with normal and MAP2-IR, and this same pattern was recapitulated in S1782E mice. These findings suggest a new conceptual framework for Sz-that a large proportion of individuals have a "MAP2opathy"-in which MAP function is altered by phosphorylation, leading to impairments of neuronal structure, synaptic protein synthesis, and function.

Effects of direct renal arterial infusion of bufalin and ouabain in conscious sheep.

1. The presence of an endogenous digitalis-like factor (EDLF) in the plasma of both normal and volume expanded animals is well documented. In this study we have used ouabain and bufalin as pharmacological analogues to mimic the renal effects of EDLF and to investigate whether any interaction occurs between atrial natriuretic factor (ANF) and EDLF. 2. Conscious Na replete sheep with chronically indwelling catheters in the renal artery received renal arterial infusion of ouabain (1000 micrograms h-1) or bufalin (500 micrograms h-1) for 60 min. Renal arterial infusion of bufalin increased sodium excretion (UNaV) from 120 +/- 13 to 596 +/- 161 mumol min-1 after 45 min. Bufalin infusion did not alter glomerular filtration rate (GFR), effective renal plasma flow (ERPF), or lithium clearance. Ouabain infusion increased UNaV from 124 +/- 57 to 764 +/- 123 mumol min-1 in the first hour after infusion. 3. ANF infusion increased UNaV from 159 +/- 34 to 583 +/- 134 mumol min-1. When renal arterial bufalin infusion was followed by renal arterial ANF infusion (50 micrograms h-1) UNaV was increased from 155 +/- 31 to 795 +/- 96 mumol min-1. This increase in UNaV is approximately equal to the sum of the separate effects of bufalin and ANF. 4. The natriuretic effects of ouabain at pharmacological doses in sheep are confirmed by this study. The data presented here do not support the hypothesis that EDLF sensitizes the kidney to the natriuretic effects of ANF.

The effect of uninephrectomy on osteocalcin metabolism in sheep: a direct evaluation of renal osteocalcin clearance.

Indirect evidence has suggested that the kidney is a major organ of clearance for osteocalcin, a circulating marker of osteoblast function. The objectives of the present study were (1) to confirm the role of the kidney in osteocalcin clearance (2) to quantify the contribution of extrarenal sites and (3) to investigate the renal mechanism(s) of osteocalcin clearance. Plasma osteocalcin levels, osteocalcin plasma clearance rate (PCR) and plasma production rate (PPR) were determined in oophorectomized (OX) and uninephrectomized oophorectomized (UOX) sheep. The osteocalcin renal extraction efficiency (REE) and the effective renal plasma flow (ERPF) were measured, and the osteocalcin renal clearance rate (RCR) was calculated. The osteocalcin PCR was reduced significantly in UOX compared with OX sheep (2.0 +/- 0.1 (n = 9) vs 2.5 +/- 0.1 litres/h (n = 44); P less than 0.0005). In UOX sheep with plasma creatinine levels less than or equal to 130 mumol/l, the osteocalcin REE was 9 +/- 1.3% and the osteocalcin RCR was 50-91% of osteocalcin PCR (n = 4). In UOX sheep with plasma creatinine levels in the range 100-440 mumol/l, there was a linear relationship between osteocalcin PCR and ERPF; the osteocalcin RCR was related to the osteocalcin PCR (RCR = 0.9 x PCR - 0.50). Intravenous infusion of the synthetic glucocorticoid triamcinolone acetonide (TA) in UOX sheep led to marked decrements in plasma osteocalcin levels and the osteocalcin PPR, and a significant increase in the osteocalcin PCR. These changes were accompanied by a 44% increase in ERPF.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of hypovolemia on the renal and cardiovascular responses to atrial natriuretic factor (ANF) infusion.

The renal and cardiovascular effects of ANF infusion have been examined in separate series of experiments; in conscious instrumented sheep following either hemorrhage (10 mL/kg body weight) or removal of 500 mL of plasma by ultrafiltration. Renal arterial infusion of hANF (99-126) at 50 micrograms/h increased sodium excretion from 99 +/- 30 to 334 +/- 102 (p less than 0.05) in normal animals, and from 77 +/- 31 to 354 +/- 118 mumol/min in hemorrhaged animals. Similarly in sheep following ultrafiltration, cardiac output and stroke volume were reduced by intravenous infusion of ANF (100 micrograms/h), although these effects were less marked than those observed in normal animals. The rapid modulation of natriuretic responses to ANF observed in volume expanded animals is not seen in this model of acute volume depletion suggesting that the mechanism through which the renal response to ANF is modulated in low sodium or volume states is not simply the reverse of that which produces rapid enhancement of response following blood volume expansion.

Comparison of the renal effects of urodilatin and atrial natriuretic factor: effect of changes in sodium status.

Urodilatin is an amino terminally extended form of atrial natriuretic factor (ANF) which is resistant to enzymatic cleavage by renal neutral endopeptidase (NEP: EC 3.4.24.11). The renal effects of infusion into the renal artery of equimolar doses of urodilatin or ANF have been compared following changes in Na status in conscious sheep. In all conditions urodilatin was a more potent natriuretic stimulus than was ANF and the natriuretic response to urodilatin was modulated by sodium status in the same way as the response to ANF: diminished by sodium depletion and enhanced by sodium loading. This study does not support the hypothesis that changes in NEP activity contribute to the modulation of the natriuretic response to ANF which is observed with modification of sodium status.

Effect of volume expansion on the natriuretic response to ouabain infusion.

Evidence exists that an endogenous substance which inhibits (Na+,K+)-ATPase, in a similar manner to the cardiac glycosides, may have important cardiovascular and renal effects. Whilst ouabain or a closely related isomer has been reported to be present in mammalian plasma, renal effects of ouabain occur only at high concentrations. The effect of expansion of blood volume on the renal response to ouabain infusion was examined in conscious sheep. Five sheep with catheters chronically implanted into the renal artery received four treatment combinations in random order: (I) vehicle (0.15 mol l-1 NaCl) infusion; (II) 500 micrograms ouabain infused into the renal artery over 60 min; (III) 500 ml 6% dextran 70 in 0.9% saline infused intravenously, and (IV) the dextran and ouabain treatments together. Treatment with either ouabain or plasma volume expansion produced modest increases in sodium excretion and urine flow. Treatment with ouabain when combined with plasma volume expansion increased sodium excretion from 82 +/- 30 to 880 +/- 203 mumol min-1 and urine flow from 1.9 +/- 1.1 to 7.5 +/- 1.6 ml min-1. This combination of treatments results in a synergistic rather than additive response. This study indicates that under some circumstances the response of the kidney to inhibition of (Na+,K+)-ATPase can be enhanced and, if inhibition can be demonstrated to occur at physiologically relevant concentrations of endogenous digitalis-like factor, would support a possible physiological role for endogenous digitalis-like factor in the regulation of sodium homeostasis.

Interaction of exogenous ouabain and chronic mineralocorticoid treatment in the kidney of the conscious sheep.

1. Ouabain is known to have natriuretic effects only at high doses, and therefore if endogenously produced ouabain has a role in the regulation of sodium excretion, the renal response to ouabain must be increased substantially in certain physiological situations. The aim of this study is to determine whether treatment with the mineralocorticoid, aldosterone, potentiates that natriuretic response to ouabain. 2. Six conscious sheep received renal arterial infusion of either vehicle or aldosterone (3 micrograms/h). Forty hours after commencement of infusion ouabain was infused into the renal artery at 400 micrograms/h for 60 min. A second infusion of ouabain was administered on the 6th day of aldosterone treatment. 3. In the absence of aldosterone, the effects on sodium excretion produced by ouabain infusion at 400 micrograms/h into the renal artery were variable and not statistically significant. Ouabain infusion after 40 h of aldosterone treatment increased sodium excretion from 40 +/- 14 to 676 +/- 69 mumol/min in the second hour following cessation of ouabain infusion (P < 0.001). Ouabain infusion after 6 days of aldosterone treatment increased sodium excretion similarly. Ouabain-stimulated sodium excretion was significantly greater during aldosterone treatment compared to vehicle treatment (P < 0.05). In contrast, no enhancement of effect was observed after acute treatment with aldosterone. 4. These results demonstrate potentiation of the natriuretic response to ouabain infusion by chronic mineralocorticoid treatment and suggest a potential role of endogenous digitalis-like factor in the physiological control of sodium homeostasis.

Natriuresis and inhibition of Na+/K(+)-ATPase: modulation of response by physiological manipulation.

1. There is considerable evidence for the existence of an endogenous inhibitor of Na+/K(+)-ATPase. The exact physiological nature and role of this postulated agent remains unclear, although it would be predicted that one of its actions would be stimulation of renal sodium excretion. 2. The natriuretic effect of renal arterial infusion of ouabain is relatively slow in onset and is sustained. 3. The natriuresis is not modified by changes in sodium status, unlike the natriuretic effect of atrial natriuretic peptide. 4. The natriuretic action of ouabain is enhanced dramatically by acute volume expansion or chronic mineralocorticoid treatment, which both result in hypokalaemia, hypertension and hypervolaemia. 5. The natriuretic response to small increments in blood pressure is markedly enhanced by treatment with ouabain. 6. We hypothesize that the interaction between the inhibition of Na+/K(+)-ATPase and elevated blood pressure could result in the shedding of sodium in conditions where there are increased levels of circulating endogenous digitalis-like factors.

Differential blockade of the renal vasoconstrictor and diuretic responses to endothelin-1 by endothelin antagonist.

1. The effect of cyclo(D-Trp-D-Asp-Pro-D-Val-Leu) (or BQ123), a selective ETA receptor antagonist, on the vasoconstrictor and diuretic responses elicited by endothelin-1 (ET-1) was examined in conscious sheep with chronic indwelling renal arterial cannulae. 2. Using low dose close renal arterial infusion, ET-1 has potent effects on the kidney causing a marked decrease in effective renal plasma flow and an increase in urine output and free water clearance in the normally hydrated animal. 3. The vasoconstrictor response to renal arterial infusion of ET-1 at 5 micrograms/h was blunted by renal arterial infusion of the ETA receptor selective antagonist, BQ123 (400 micrograms/h). 4. In contrast, the effect of ET-1 on urine production and free water clearance was not affected by this dose of BQ123. 5. The differential effect of BQ123 on renal blood flow and urine production suggests that these effects of endothelin on the kidney are mediated through different receptor mechanisms.

Renal effects of atrial natriuretic factor (99-126) in water-deprived sheep.

1. The renal response to renal arterial infusion of synthetic human atrial natriuretic factor (ANF) (99-126) at 50 micrograms/h was examined in conscious sheep dehydrated by 48 h water deprivation and was compared with the response of normally hydrated animals. 2. Renal arterial infusion of ANF produced increases in the excretion of Na, K, Ca and urine in both dehydrated and normally hydrated animals, although the effect was significantly blunted in dehydrated animals compared with normally hydrated animals. 3. The attenuation of renal effects of ANF in dehydrated animals is probably due to the negative sodium and/or fluid balance of the dehydrated animals.

Renal effects of atrial natriuretic factor (99-126) in potassium loaded sheep.

1. The renal response to renal arterial infusion of synthetic atrial natriuretic factor (ANF 99-126) was examined in conscious sheep following dietary K loading, and compared with the response in normal sheep. 2. Renal arterial infusion of ANF in K loaded sheep increased the excretion of Na and Ca, but did not affect the excretion of K. 3. The natriuretic effect of ANF was attenuated in K loaded animals, possibly as a consequence of the reduction in Na status which is associated with K loading.

Renal effects of atrial natriuretic factor (99-126) in conscious sodium-replete sheep.

1. The effect of renal arterial infusion of synthetic human atrial natriuretic factor (ANF (99-126] on renal function in the conscious euvolaemic sheep was characterized. ANF (99-126) was infused for 2 h at 5 and 50 micrograms/h into the renal artery of crossbred Merino ewes with chronically indwelling cannulae inserted in the renal artery. The effect on absolute and fractional excretion of Na, K, Ca, Cl and HCO3, glomerular filtration rate (GFR), effective renal plasma flow (ERPF) and free water clearance (CH2O) were measured. 2. Infusion at 50 micrograms/h produced a fourfold increase in Na and Cl excretion. Ca excretion increased eightfold, while K and HCO3 increased by small amounts. At the lower dose only Na, Cl and Ca excretion increased significantly. The changes in absolute excretion of each ion were closely mirrored by changes in fractional excretion. CH2O became more negative at both levels of infusion. Small changes in GFR were measured at both rates of infusion. No changes in ERPF or renin secretion were observed. 3. ANF (99-126) infusion at 50 micrograms/h for 1 h increased the excretion of Li, such that more than 70% of the change in Na excretion was associated with the changes in Li clearance. Changes in GFR accounted for less than 10% of change in Na excretion. 4. Following either long-term (50 micrograms/h for 6 h) or repeated short-term (20 micrograms/h for 30 min) infusions of ANF (99-126), the response of Na excretion was not sustained. The mechanisms of the tachyphylaxis remains undetermined. 5. ANF (99-126) is a powerful stimulus to the absolute and fractional excretion of Na, K, Ca, Cl and HCO3. The mechanism of action is not known, but appears to be related to changes in tubular function and/or a change in glomerulotubular balance.

Renal actions of atrial natriuretic factor: modulation of effect by changes in sodium status and aldosterone.

To examine the effect of changes in Na status on the renal response to atrial natriuretic factor (ANF), human ANF-(99-126) was infused into the renal artery of conscious sheep while Na replete, Na depleted, or Na loaded. The natriuretic response to ANF was attenuated during Na depletion and enhanced in Na-loaded animals. To demonstrate that the enhancement or attenuation of response was related to Na status rather than to the initial level of Na excretion, aldosterone was infused into the renal artery for 2 h to decrease Na excretion to a level inappropriately low for the animal's Na status (and not different from Na-depleted animals), and they were again challenged with ANF. Their response to ANF, however, was not significantly different from that in normal Na-replete animals but was significantly greater than that observed in Na-depleted animals. Similarly, Na-loaded animals treated with aldosterone had control Na excretion in the Na-replete range; however, their response to ANF was not significantly different from that of Na-loaded animals. The response to ANF was enhanced in sheep treated with aldosterone for 48 h, consistent with Na retention and hypervolemia, secondary to aldosterone treatment. The study demonstrates that Na status or some associated physiological parameter is an important determinant of the natriuretic response to ANF. The present series of experiments demonstrate that changes in aldosterone levels per se are not a determinant of the natriuretic response to ANF.

Dendrimer-supported combinatorial chemistry.

A new methodology for the construction of combinatorial libraries is described. The approach, termed dendrimer-supported combinatorial chemistry (DCC), centers on the use of dendrimers as soluble supports. Salient features of DCC include solution phase chemistry, homogeneous purification, routine characterization of intermediates, and high support loadings. To demonstrate the feasibility of DCC, single compounds and a small combinatorial library were prepared via the Fischer indole synthesis. Excellent product yields and purities were obtained, and dendrimer-protected intermediates could be routinely analyzed by 1H and 13C NMR and by mass spectrometry. The results indicate that DCC is a general and efficient strategy for the generation of combinatorial libraries.

Processing and bioactivity of the corpuscles of Stannius protein of the Australian eel.

The primary structure of the major protein from the Corpuscles of Stannius (CS) of the Australian eel was elucidated from the cDNA sequence and was found to bear close similarity to the N-terminal amino acid sequence of the presumably homologous salmon hormone, teleocalcin (TC). The cDNA sequence predicted a preproprotein of 263 amino acids. Following removal of a 17 amino acid signal peptide, specific monobasic cleavage at an Arg-Phe bond generates the 231 amino acid mature form of the protein. The isolation and sequence determination of the prosequence confirms that the precursor contains a prosegment of 15 residues. Various fragments of the protein have been synthesized chemically and their biological activity assessed. The N-terminal 1-20 fragment of the mature protein inhibits calcium uptake in fingerling trout, the effect being similar, but not equipotent to salmon teleocalcin. Further, infusion of either the N-terminal 1-20 or the 81-94 fragment at 50 µg/h into the renal artery of conscious sheep, caused significant decreases in systemic plasma potassium concentration and in potassium excretion. The 1-20 fragment also gave rise to a small but significant increase in sodium excretion. Infusion of TC at the same rate results in a significant decrease in plasma potassium and phosphate concentration as well as a significant decrease in potassium excretion. Bovine PTH (1-34) at 100 µg/h causes a small decrease in plasma potassium and phosphate and an increase in plasma calcium concentration, and was the only peptide to cause a significant decrease in calcium excretion.