Brain-derived adrenomedullin controls blood volume through the regulation of arginine vasopressin production and release

Central nervous system-derived adrenomedullin (AM) has been shown to be a physiological regulator of thirst. Administration of AM into the lateral ventricle of the brain attenuated water intake, whereas a decrease in endogenous AM, induced by an AM-specific ribozyme, led to exaggerated water intake. We hypothesized that central AM may control fluid homeostasis, in part by regulating plasma arginine vasopressin (AVP) levels. To test this hypothesis, AM or a ribozyme specific to AM was administered intracerebroventricularly, and alterations in plasma AVP concentrations were examined under basal and stimulated (hypovolemic) conditions. Additionally, we examined changes in blood volume, kidney function, and plasma electrolyte and protein levels, as well as changes in plasma aldosterone concentrations. Intracerebroventricular administration of AM increased plasma AVP levels, whereas AM ribozyme treatment led to decreased plasma AVP levels under stimulated conditions. During hypovolemic challenges, AM ribozyme treatment led to an increased loss of plasma volume compared with control animals. Although overall plasma osmolality did not differ between treatment groups during hypovolemia, aldosterone levels were significantly higher and, consequently, plasma potassium concentrations were lower in AM ribozyme-treated rats than in controls. These data suggest that brain-derived AM is a physiological regulator of vasopressin secretion and, thereby, fluid homeostasis.     

Intermedin/adrenomedullin-2 acts within central nervous system to elevate blood pressure and inhibit food and water intake

Intermedin (IMD)/adrenomedullin-2 (AM2) is a novel peptide that was independently discovered by two groups. The 47-amino acid peptide is homologous to adrenomedullin (AM) and can activate both the AM and calcitonin gene-related peptide (CGRP) receptors. IMD should therefore have actions similar to those of AM and CGRP. Indeed, like AM and CGRP, intravenous administration of IMD decreased blood pressure in rats and mice. We demonstrate here that immunoreactive IMD is present in plasma as well as heart, lung, stomach, kidney, pituitary, and brain. Because IMD is present in brain and both AM and CGRP have potent central nervous system (CNS) effects, we examined the ability of IMD within brain to regulate blood pressure and ingestive behaviors. Administration of IMD into the lateral cerebroventricle of rats caused significant, long-lasting elevations in mean arterial pressure and heart rate. These elevations are similar to the effects of CGRP and significantly greater than the effects of AM. IMD-induced elevations in mean arterial pressure were inhibited by intravenous administration of phentolamine, indicating that IMD activates the sympathetic nervous system. Intracerebroventricular administration of IMD also inhibited food and water intake in sated and in food- and water-restricted animals. The effects on feeding are likely related to activation of the CGRP receptor and are independent of the effects on water intake, which are likely through the AM receptor. Our data indicate that IMD has potent actions within the CNS that may be a result of the combined activation of both AM and CGRP receptors.     

Different responses of atrial natriuretic peptide secretion and its receptor density to salt intake in rats

This study investigated whether high-salt intake influences atrial natriuretic peptide (ANP) system, atrial content, and release rate of ANP as well as receptor density in the kidney were measured in salt intake rats. Male Sprague-Dawley rats received either 0.9% or 2% salt in their drinking water for 10 days. The stretch-induced ANP secretion from isolated perfused non-beating left atria was accentuated, and the production of cGMP by ANP in renal cortical tissue membranes were pronounced in rats exposed to 0.9% salt for 10 days but not in rats exposed to 2% salt. The levels of ANP receptor density and expression in renal cortex were decreased in 2% salt intake rats but not in 0.9% salt intake rats. No significant differences in atrial and plasma concentrations of ANP and water balance were observed in both salt intakes. Therefore, these results suggest that atrial ANP secretion and its binding sites in the kidney may respond differently to ingested salt concentrations in rats.     

肾大部切除、高盐摄食对SD大鼠脑心房钠尿肽的影响

为探讨脑内心房钠尿肽（ＡＮＰ）的作用,本工作采用ＳＤ大鼠,用放射免疫方法测定３／４肾大部切除与高盐摄食后脑内ＡＮＰ的含量。结果表明,对照组大鼠脑内ＡＮＰ分布广泛。３／４肾切除大鼠每日饮水量,尿量均比对照组高（Ｐ＜０．０５）,尿钠浓度低于对照组时,脑内ＡＮＰ含量尽管略有增加,但１０个核团（下丘脑室周核、弓状核、室旁核、视前室周核、中缝背核、尾壳核、杏仁核、脑桥背侧部、蓝斑和大脑皮质）ＡＮＰ含量和对照组无明显差别（Ｐ＞０．０５）。高盐摄食组每日饮水量和尿量均比对照组高,且尿钠浓度高于对照组,同时下丘脑室周核和弓状核ＡＮＰ含量比对照组高（Ｐ＜０．０５）。上述结果提示,大脑第三脑室前腹侧区（ＡＶ３Ｖ区）的ＡＮＰ可能在水盐调节上发挥重要作用。     

Effect of high and low sodium intake on urinary aquaporin-2 excretion in healthy humans

The degree of water transport via aquaporin-2 (AQP2) water channels in renal collecting duct principal cells is reflected by the level of the urinary excretion of AQP2 (u-AQP2). In rats, the AQP2 expression varies with sodium intake. In humans, the effect of sodium intake on u-AQP2 and the underlying mechanisms have not previously been studied. We measured the effect of 4 days of high sodium (HS) intake (300 mmol sodium/day; 17.5 g salt/day) and 4 days of low sodium (LS) intake (30 mmol sodium/day; 1.8 g salt/day) on u-AQP2, fractional sodium excretion (FE(Na)), free water clearance (C(H2O)), urinary excretion of PGE(2) (u-PGE(2)) and cAMP (u-cAMP), and plasma concentrations of vasopressin (AVP), renin (PRC), ANG II, aldosterone (Aldo), atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) in a randomized, crossover study of 21 healthy subjects, during 24-h urine collection and after hypertonic saline infusion. The 24-h urinary sodium excretion was significantly higher during HS intake (213 vs. 41 mmol/24 h). ANP and BNP were significantly lower and PRC, ANG II, and Aldo were significantly higher during LS intake. AVP, u-cAMP, and u-PGE(2) were similar during HS and LS intake, but u-AQP2 was significantly higher during HS intake. The increases in AVP and u-AQP2 in response to hypertonic saline infusion were similar during HS and LS intake. In conclusion, u-AQP2 was increased during HS intake, indicating that water transport via AQP2 was increased. The effect was mediated by an unknown AVP-independent mechanism.     

Salt Appetite Is Reduced by a Single Experience of Drinking Hypertonic Saline in the Adult Rat

Salt appetite, the primordial instinct to favorably ingest salty substances, represents a vital evolutionary important drive to successfully maintain body fluid and electrolyte homeostasis. This innate instinct was shown here in Sprague-Dawley rats by increased ingestion of isotonic saline (IS) over water in fluid intake tests. However, this appetitive stimulus was fundamentally transformed into a powerfully aversive one by increasing the salt content of drinking fluid from IS to hypertonic saline (2% w/v NaCl, HS) in intake tests. Rats ingested HS similar to IS when given no choice in one-bottle tests and previous studies have indicated that this may modify salt appetite. We thus investigated if a single 24 h experience of ingesting IS or HS, dehydration (DH) or 4% high salt food (HSD) altered salt preference. Here we show that 24 h of ingesting IS and HS solutions, but not DH or HSD, robustly transformed salt appetite in rats when tested 7 days and 35 days later. Using two-bottle tests rats previously exposed to IS preferred neither IS or water, whereas rats exposed to HS showed aversion to IS. Responses to sweet solutions (1% sucrose) were not different in two-bottle tests with water, suggesting that salt was the primary aversive taste pathway recruited in this model. Inducing thirst by subcutaneous administration of angiotensin II did not overcome this salt aversion. We hypothesised that this behavior results from altered gene expression in brain structures important in thirst and salt appetite. Thus we also report here lasting changes in mRNAs for markers of neuronal activity, peptide hormones and neuronal plasticity in supraoptic and paraventricular nuclei of the hypothalamus following rehydration after both DH and HS. These results indicate that a single experience of drinking HS is a memorable one, with long-term changes in gene expression accompanying this aversion to salty solutions.     

High salt intake attenuates the development of hypertension in two-kidney, one-clip Goldblatt rats.

Effects of high salt intake on the early onset of hypertension were examined in two-kidney, one-clip rats. They were divided into high salt and control groups which were supplied with 1.0% NaCl and tap water, respectively, as a drinking solution for 12 days after clipping the left renal artery. The high salt group showed a lower plasma renin concentration and a higher plasma atrial natriuretic peptide (ANP) along with an attenuation of the magnitude of early hypertension, as compared with the control group. A significant positive correlation between blood pressure and plasma renin concentration and an inverse correlation between plasma renin concentration and ANP were shown. Cortical renal renin content was comparable between the two groups. In another two groups of sham-clipped rats, the high salt group did not differ from the tap water-drinking group in any of the parameters examined, except that ANP was significantly higher. These results demonstrate that high salt intake attenuates the developmental phase of hypertension in two-kidney, one-clip rats by increasing the ANP and suppressing the release of renin.     

盐胁迫下丛枝菌根真菌对玉米水分和养分状况的影响

在ＮａＣｌ胁迫下无论接种ＡＭ真菌与否玉米植株生物产量均减少,但不接种处理的减少幅度比较种处理的高１０个百分点左右,盐胁迫下接种ＡＭ真菌的玉米根系和地上部的干重、叶片水热均高于不接种处理、叶片脯氨酸含量低于不接种处理,在盐胁迫下真菌菌丝对玉米植株营养的贡献由４５．３％降为４２．６％,ＡＭ真菌对植株生长的效应反而由３０．９％提高到６３．５％,说明ＡＭ真菌主米耐盐性的机理与改善植株的水分状况和Ｐ营养状况     

Adrenomedullin inhibits prostate cancer cell proliferation through a cAMP-independent autocrine mechanism

Adrenomedullin (AM) is a multifunctional peptide expressed in the normal and malignant prostate, and in prostate cancer cells. To elucidate the potential role of AM in prostate cancer, we have transfected the human AM gene into PC-3, DU 145, and LNCaP prostate cancer cells. Northern blot, Western blot, and radioimmunoassay techniques confirmed an increase in the synthesis and secretion of the 6kDa mature peptide, in the AM-transfected clones. Proliferation and cell cycle assays demonstrated that AM overexpression inhibited cell proliferation in PC-3 and LNCaP cells through a G0/G1 cell cycle arrest, but not in DU 145 cells. In vivo growth assays also confirmed that, at least in PC-3, AM produced a very significant reduction of tumor volume. In addition, the three cell lines expressed the CL/RCP/RAMP-2 receptor complex by RT-PCR, which suggests that AM peptide acts through an autocrine loop in prostate cancer cells. Although cAMP elevation is the most common pathway involved in AM signalling, stimulation of PC-3, DU 145, and LNCaP with synthetic AM did not increase intracellular cAMP. However, short-term stimulation of PC-3 cells with synthetic AM increased ERK1/2 activation. On the contrary, long-term stimulation, or AM overexpression, caused a reduction in the basal activation of ERK1/2. In summary, our results demonstrate that AM (either overexpressed or exogenously added) causes an inhibition of prostate cancer cell growth. This inhibition does not depend on changes in intracellular cAMP levels, but may be related to ERK1/2 activation.     

Inhibition of dehydration-induced water intake by glucocorticoids is associated with activation of hypothalamic natriuretic peptide receptor-A in rat

Atrial natriuretic peptide (ANP) provides a potent defense mechanism against volume overload in mammals. Its primary receptor, natriuretic peptide receptor-A (NPR-A), is localized mostly in the kidney, but also is found in hypothalamic areas involved in body fluid volume regulation. Acute glucocorticoid administration produces potent diuresis and natriuresis, possibly by acting in the renal natriuretic peptide system. However, chronic glucocorticoid administration attenuates renal water and sodium excretion. The precise mechanism underlying this paradoxical phenomenon is unclear. We assume that chronic glucocorticoid administration may activate natriuretic peptide system in hypothalamus, and cause volume depletion by inhibiting dehydration-induced water intake. Volume depletion, in turn, compromises renal water excretion. To test this postulation, we determined the effect of dexamethasone on dehydration-induced water intake and assessed the expression of NPR-A in the hypothalamus. The rats were deprived of water for 24 hours to have dehydrated status. Prior to free access to water, the water-deprived rats were pretreated with dexamethasone or vehicle. Urinary volume and water intake were monitored. We found that dexamethasone pretreatment not only produced potent diuresis, but dramatically inhibited the dehydration-induced water intake. Western blotting analysis showed the expression of NPR-A in the hypothalamus was dramatically upregulated by dexamethasone. Consequently, cyclic guanosine monophosphate (the second messenger for the ANP) content in the hypothalamus was remarkably increased. The inhibitory effect of dexamethasone on water intake presented in a time- and dose-dependent manner, which emerged at least after 18-hour dexamethasone pretreatment. This effect was glucocorticoid receptor (GR) mediated and was abolished by GR antagonist RU486. These results indicated a possible physiologic role for glucocorticoids in the hypothalamic control of water intake and revealed that the glucocorticoids can act centrally, as well as peripherally, to assist in the normalization of extracellular fluid volume.     

Adaptation capacity of Boer goats to saline drinking water

Due to global climatic changes, water and soil salinization is an increasing worldwide phenomenon, thus creating new threats for farm animal production. The present study was designed to investigate the adaptation capacity of goats towards sodium chloride (NaCl) in drinking water. Twelve non-pregnant Boer goats with an average body weight of 50.5 ± 9.0 kg were kept in individual pens. The study was conducted in four phases applying a two-choice preference test. In the control phase (phase 1) only fresh water was supplied in two containers. In phase 2, water with different salt concentrations (0.25%, 0.5%, 0.75%, 1.0%, 1.25% and 1.5%) was offered in one container and tap water in the other (sensitivity test). During the third phase (adaptation), goats were stepwise habituated to saline water by offering only saline water with different increasing concentrations (between 0% and 1.5% NaCl) in both containers. Subsequently, in phase 4 (sensitivity re-test) the same treatment as in phase 2 was repeated. Goats had ad libitum access to hay, water and a mineral licking block. Individual water and feed intake were recorded daily, while body weight and body condition score were measured every 2^nd week. Body weight was not affected by saline water intake, whereas dry matter intake and body condition scores decreased significantly during the experiment. Water intake was significantly (P<0.001) higher in phase 2 (sensitivity test) and phase 3 (adaptation), compared to phase 1 (control) and phase 4 (sensitivity re-test). Total sodium intake followed the same pattern. In phase 2, when goats had the choice between fresh and saline water for the first time they preferred higher salt concentrations and consumed significantly (P<0.001) higher amounts of saline water (75.4 ± 53.2 g/kg BW^0.82 per day) than in the re-test (40.4 ± 34.0 g/kg BW^0.82 per day) after the habituation period. Thus, salt discrimination rejection thresholds were lowered to 1.25% in phase 4 compared to 1.5% in phase 2. The results suggest that a stepwise adaptation to saline drinking water in goats is an effective method to habituate the animals to saline water intake when concentrations were below 1.5%. Goats reacted more sensitively to the salinity of drinking water after prolonged exposure to saline water indicating flexible regulation mechanisms depending on the total sodium balance of the animal.     

Decrease of TSH levels and epithelium/colloid ratio in rat thyroid glands following administration of proadrenomedullin N-terminal peptide (12-20).

Adrenomedullin (AM) exerts a potent and long-lasting hypotensive effect and is considered to be an important hormone in blood pressure control. AM is a 52-amino-acid peptide synthesized as part of a 185-amino-acid preprohormone that also contains 20-amino-acid residues in the N-terminus, which has similar biological activity. This sequence is named a proadrenomedullin N-terminal 20 peptide (PAMP). Also, proadrenomedullin N-terminal peptide (PAMP)(12-20) exerts vasodepressor response, however this response is 3-fold less potent than the effect evoked by full-sequence peptide. Both AM and PAMP controls secretory activity of the pituitary gland and adrenal cortex, however, their action on the other endocrine glands is not recognized. Therefore, the aim of the study was to examine whether PAMP(12-20) is able to affect the structure and function of the rat thyroid gland. In adult female rats, subcutaneous PAMP(12-20) administration (1 or 4 nmol/rat/day for 6 days, autopsy 60 min after the last injection) had no effect on the weight of the thyroid gland. Peptide administration however, resulted in a dose-dependent increase in the volume of thyroid colloid, and lowered epithelium/colloid ratio in the gland (3.76 +/- 0.49, 2.66 +/- 0.27, 2.38 +/- 0.26, means +/- SE, n = 6, control, 1 and 4nmol PAMP/rat, respectively). PAMP administration changed neither the length of thyroid capillaries per unit area of surface nor their diameter. Lower dose of PAMP(12-20) significantly lowered blood TSH concentration (p < 0.01) while total and free T3 and T4 concentrations remained unchanged. Collectively, these findings suggest that PAMP(12-20) exerts a mild inhibitory effect on secretory activity of the rat thyroid gland.     

心房钠尿肽以血管紧张素及禁水引起大鼠饮水行为的影响

Atrial natriuretic peptide (ANP) present in the brain has been reported to have profound effects on water and salt metabolism. This study was designed to observe the effect of intracerebroventricular (ICV) injection of ANP on drinking behavior of rats, induced by centrally administered angiotensin II (Ang II) and 24-hours water deprivation, by using a T-maze to measure the speed they ran in a runway for water rewards. In 24-hours water deprived rats ICV injection of ANP resulted in a significant decrease of either running speed or water intake. Drinking behavior induced by ICV injection of Ang II in normally hydrated rats was also significantly inhibited by a prior injection of ANP. These findings suggest that ANP in the brain plays an important role in the central control of drinking behavior.     

Adrenomedullin receptors: molecular identity and function.

Since its discovery in 1993 adrenomedullin (AM) has been the subject over 600 published articles. This multifunctional peptide has powerful vasodilator actions and recent evidence from AM gene-deleted mice suggest that AM plays an essential role in vascular development. However the lack of valid AM receptor clones and non-peptide receptor ligands has considerably slowed research progress on this important peptide. In this review we have focused on the proposition that the calcitonin receptor-like receptor (CRLR) is a receptor both for AM and the related vasoactive peptide calcitonin gene-related peptide (CGRP). The receptor activity modifying proteins (RAMPs) that are essential for defining CRLR pharmacology will also be discussed. We will describe how AM receptors have been reported to signal and be regulated and to consider whether further receptors for AM beyond CRLR/RAMP combinations might exist.     

Adrenomedullin inhibits angiotensin AT1A receptor expression and function in cardiac fibroblasts

Adrenomedullin (AM) is a multifunctional peptide hormone with wide-ranging actions related to cardiovascular homeostasis. AM receptors are highly expressed in the heart and AM has antihypertrophic and antiproliferative effects on cardiac myocytes and fibroblasts, respectively. We have investigated the interaction between AM and angiotensin II (Ang II) signalling in neonatal cardiac fibroblast cultures to determine whether the antagonistic effects of AM are mediated via the modulation of Ang II receptors. Cardiac fibroblasts exclusively expressed the Ang II type 1 receptor (AT(1)R) and binding to this site was downregulated by 35% following an 18-h incubation with 100 nM AM. Levels of AT(1A)R mRNA were dose-dependently lowered by AM, with a maximal 40-50% inhibition by 6 h. The decreases in both AT(1)R binding and AT(1A)R mRNA levels were mimicked by 8-Br-cAMP or forskolin, suggesting that the effects of AM were mediated via an elevation of cAMP. In cardiac fibroblasts pretreated with AM, the Ang II induction of collagen biosynthesis was attenuated, although basal collagen synthesis was unaffected. These data suggest that AM mediates the heterologous downregulation of AT(1)R expression via a relatively rapid decrease in AT(1A)R mRNA pools. This interaction may represent a relevant pathophysiological mechanism for modulating Ang II responsiveness in the diseased heart.     

Effect of sodium chloride on bakers' yeast growing in gelatin

In recent years, industrial fermentation researchers have shifted their attention from liquid to solid and semisolid culture conditions. We converted liquid cultures to the semisolid mode by adding high levels of gelatin. Previous studies on liquid cultures have revealed the inhibitory activity of mineral salts, such as NaCl, on the fermentation of sugars by yeasts. We made a kinetic study of the effects of 1 to 5% (wt/vol) NaCl on the alcoholic fermentations of glucose by Saccharomyces cerevisiae in a growth medium containing 16% gelatin. Our results showed that the effect of high salt content on semisolid culture is essentially the same as the effect on liquid culture; i.e., as the salt content increased, the following occurred: (i) the growth of yeasts decreased, (ii) the lag period of the yeast biomass curve lengthened, (iii) the sugar intake was lowered, (iv) the yield of ethanol was reduced, and (v) the production of glycerol was increased. We observed a new relationship correlating the area of kinetic hysteresis with ethanol production rate, acetaldehyde concentration, and the initial NaCl concentration.     

Influence of arbuscular mycorrhizae on photosynthesis and water status of maize plants under salt stress

The influence of arbuscular mycorrhizal (AM) fungus Glomus mosseae on characteristics of the growth, water status, chlorophyll concentration, gas exchange, and chlorophyll fluorescence of maize plants under salt stress was studied in the greenhouse. Maize plants were grown in sand and soil mixture with five NaCl levels (0, 0.5, 1.0, 1.5, and 2.0 g/kg dry substrate) for 55 days, following 15 days of non-saline pretreatment. Under salt stress, mycorrhizal maize plants had higher dry weight of shoot and root, higher relative chlorophyll content, better water status (decreased water saturation deficit, increased water use efficiency, and relative water content), higher gas exchange capacity (increased photosynthetic rate, stomatal conductance and transpiration rate, and decreased intercellular CO(2) concentration), higher non-photochemistry efficiency [increased non-photochemical quenching values (NPQ)], and higher photochemistry efficiency [increased the maximum quantum yield in the dark-adapted state (Fv/Fm), the maximum quantum yield in the light-adapted sate (Fv'/Fm'), the actual quantum yield in the light-adapted steady state (varphiPSII) and the photochemical quenching values (qP)], compared with non-mycorrhizal maize plants. In addition, AM symbiosis could trigger the regulation of the energy biturcation between photochemical and non-photochemical events reflected in the deexcitation rate constants (kN, kN', kP, and kP'). All the results show that G. mosseae alleviates the deleterious effect of salt stress on plant growth, through improving plant water status, chlorophyll concentration, and photosynthetic capacity, while the influence of AM symbiosis on photosynthetic capacity of maize plants can be indirectly affected by soil salinity and mycorrhizae-mediated enhancement of water status, but not by the mycorrhizae-mediated enhancement of chlorophyll concentration and plant biomass.     

Regulation of salt gland, gut and kidney interactions

Marine birds can drink seawater because their cephalic 'salt' glands secrete a sodium chloride (NaCl) solution more concentrated than seawater. Salt gland secretion generates osmotically free water that sustains their other physiological processes. Acclimation to saline induces interstitial water and Na move into cells. When the bird drinks seawater, Na enters the plasma from the gut and plasma osmolality (Osm(pl)) increases. This induces water to move out cells expanding the extracellular fluid volume (ECFV). Both increases in Osm(pl) and ECFV stimulate salt gland secretion. The augmented intracellular fluid content should allow more rapid expansion of ECFV in response to elevated Osm(pl) and facilitate activation of salt gland secretion. To fully utilize the potential of the salt glands, intestinally absorbed NaCl must be reabsorbed by the kidneys. Thus, Na uptake at gut and renal levels may constrain extrarenal NaCl secretion. High NaCl intake elevates plasma aldosterone concentration of Pekin ducks and aldosterone stimulates intestinal and renal water and sodium uptake. High NaCl intake induces lengthening of the small intestine of adult Mallards, especially males. High NaCl intake has little effect on glomerular filtration rate or tubular sodium Na uptake of birds with competent salt glands. Relative to body mass, kidney mass and glomerular filtration rate (GFR) are greater in birds with salt glands than in birds that do not have them. Birds with salt glands do not change GFR, when they drink saline. Thus, their renal filtrate contains excess Na that is, in some species, almost completely renally reabsorbed and excreted in a more concentrated salt gland secretion. Na reabsorption by kidneys of other species, like mallards is less complete and their salt glands make less concentrated secretion. Such species may reflux urine into the hindgut, where additional Na may also be reabsorbed for extrarenal secretion. During exposure to saline, marine birds maintain elevated aldosterone levels despite high Na intake. Marine birds are excellent examples of physiological plasticity.     

Calcium(II)-dependent catalytic activity of the Azoarcus ribozyme: testing the limits of resolution for in vitro selection

Group I intron ribozymes isolated from natural sources have a strict dependence on the divalent metal cations Mg(II) or Mn(II) for catalytic activity. However, mutant versions of the Tetrahymena ribozyme have been previously isolated in the laboratory that show demonstrable activity in 10 mM CaCl(2) as the only supplied salt. Here, we sought to discover similar variants of another group I intron that is likely more evolutionarily specialized. We used in vitro selection to isolate a Ca(II)-dependent variant of the naturally-occurring form of the Azoarcus ribozyme, which is half the size of the Tetrahymena ribozyme and possesses an extremely high G+C content of 71%. A mutation of G to A at position 118 was selected in multiple independent trials. Activity of the mutant is very poor in Ca(II) and can only be observed after RT-PCR, highlighting the power of in vitro selection to isolate molecules with rare and low-level activities. The mutation likely confers an alternate but rare folded conformation that permits accommodation of Ca(II) ions and catalysis. This work also serves to caution that although a selection may be successful, isolates may not be catalytically proficient enough to provide useful levels of activity.