Biomedical subjects

E Gylfe

Publications and source records attributed to E Gylfe.

At least 37 records · Page 2Linked to original sources

PubMed1995

Effects of the antihypercalcemic drugs gallium nitrate and pamidronate on hormone release of pathologic human parathyroid cells.

BACKGROUND: Gallium nitrate and the bisphosphonates pamidronate and its dimethylated derivative comprise antihypercalcemic drugs with actions on bone. This study examines the in vitro effects of these compounds on human parathyroid cells. METHODS: Parathyroid hormone (PTH) release and the concentration of cytoplasmic calcium ion (Ca2+) of dispersed cells from parathyroid glands of 27 patients with sporadic primary or uremic hyperparathyroidism was measured. RESULTS: In 1.25 mmol/L external Ca2+, 200 mumol/L gallium nitrate inhibited PTH release from preparations of primary and uremic hyperparathyroidism by 14% and 22%, respectively. Similar reductions were evident also in 0.5 and 3.0 mmol/L Ca2+. The gallium nitrate-induced suppression of PTH release was dose dependent in the 2 to 200 mumol/L range. Cytoplasmic Ca2+ concentration displayed a biphasic rise on elevation of external Ca2+ and remained unaffected by gallium nitrate. None of the bisphosphonates altered PTH release of pathologic human or normal bovine parathyroid cells. CONCLUSIONS: The results support clinical usefulness of gallium nitrate through its dual actions on bone and the parathyroid. The findings substantiate that gallium may reduce PTH release by stabilization of the plasma membrane rather than by interference with the surface cation receptor mediating Ca2+ regulation of the secretion.

Adult↗

PubMed1994

Variations in ATP-sensitive K+ channel activity provide evidence for inherent metabolic oscillations in pancreatic beta-cells.

The cell-attached configuration of the patch clamp technique was used for studying slow variations in the activity of the ATP-sensitive K+ channels in pancreatic beta-cells isolated from mouse and man. In 0 or 3 mM glucose, the fraction of time the channels were open exhibited oscillations with frequencies in the 0.25-0.40/min range. This phenomenon is a strong argument for inherent fluctuations in the ATP production of the beta-cells. Variations in metabolism may thus be a major determinant for the characteristic large amplitude oscillations of cytoplasmic Ca2+ with equivalent frequency.

Action Potentials↗

PubMed1994

Rapid down-regulation of substance P binding to guinea-pig pancreatic acinar cells during homologous desensitization.

Binding of 125I-labelled peptides, cytoplasmic Ca2+ concentration ([Ca2+]i) and amylase release were studied in guinea-pig pancreatic acinar cells during exposure to substance P (SP), and cholecystokinin octapeptide (CCK-8). Pre-incubation of cells at 22 degrees C with 0.03 nM to 1 microM SP for 10 min or at 37 degrees C for 5 min followed by acid or neutral washes reduced subsequent binding of 125I-Bolton-Hunter reagent-labelled SP (125I-BH-SP) in a biphasic manner by up to 95%. Incubation at 4 degrees C eliminated high-affinity binding of 125I-BH-SP and concentrations of SP above 1 nM were required for inhibition of subsequent tracer binding. Pre-incubation of cells at 37 degrees C with 1 nM to 1 microM CCK-8 for 10 min followed by neutral washes reduced subsequent binding of 125I-BH-CCK-8 by up to 65%. In cell suspensions, the [Ca2+]i response to SP was gradually reduced by pre-exposure to increasing agonist concentrations from 0.2 to 20 nM. Pre-incubation with high SP concentrations for 10 min caused profound reduction of subsequent amylase responses to SP, whereas secretion was little affected in corresponding experiments with CCK-8. Down-regulation of receptor binding is not important during short exposure to CCK-8, but it is a pronounced and rapid phenomenon during SP exposure, which explains tachyphylaxis of [Ca2+]i and amylase responses.

Amylases↗

PubMed1994

Synchronous oscillations of cytoplasmic Ca2+ and insulin release in glucose-stimulated pancreatic islets.

The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in single pancreatic mouse islets superfused in a system allowing concomitant recordings of insulin release. When glucose was raised from 3 to 11 mM, [Ca2+]i responded by a transient lowering followed by a rise to an average level of 192 +/- 11 nM. In 77% of the islets the rise was associated with the gradual appearance of oscillations, which were either fast (2-7/min), slow (0.3-0.9/min), or a combination of both types. The characteristics of the fast [Ca2+]i oscillations were those expected from a relationship with the electrical burst activity in islets. Accordingly, in most cases the fast oscillations were remarkably regular. The slow [Ca2+]i oscillations had characteristics similar to the large amplitude ones in individual beta-cells. Whereas glucagon and dibutyryl cAMP could transform slow islet oscillations into fast ones, the alpha 2-adrenergic agonist clonidine had the opposite effect. The rapid islet oscillations were also facilitated by elevated concentrations of extracellular Ca2+. Reinforcing the arguments for [Ca2+]i oscillations as responsible for a pulsatile insulin secretion it was possible to demonstrate that the release of the hormone from single islets is synchronized with the slow [Ca2+]i oscillations.

Animals↗

PubMed1994

Calcium sensing by human medullary thyroid carcinoma cells.

Regulation of the cytoplasmic calcium concentration ([Ca2+]i) was studied in fura-2-loaded C-cells from two human medullary thyroid carcinomas (MTC). K+ depolarization induced sustained rise of [Ca2+]i reversed by verapamil. Elevation of external Ca2+ from 0.5 to 3.0 mM triggered regular oscillations or steady-state increases of [Ca2+]i. In Ca(2+)-deficient medium Sr2+ caused steady-state increase or oscillations of the 340/380 nm fluorescence ratio. The Ca2+ and Sr2+ actions were partially reversible by verapamil. La3+ and Ce3+ elicited transient [Ca2+]i peaks independent of external Ca2+, but no oscillations. The results indicate that human MTC cells express a parathyroid-like Ca2+ sensor coupled to intracellular mobilization and influx of Ca2+. A voltage-dependent Ca2+ influx may be of importance for the oscillations of [Ca2+]i.

Calcium↗

PubMed1994

Glucose-induced oscillations of Ba2+ in pancreatic beta-cells occur without involvement of intracellular mobilization.

Ba2+ was used as a substitute for Ca2+ in analyzing the mechanisms responsible for glucose-induced Ca2+ oscillations in pancreatic beta-cells. The 340/380-nm fluorescence excitation ratio was recorded in individual mouse beta-cells loaded with the indicator fura-2. In 3 mM glucose, Ba2+ entered the cell in a concentration-dependent manner and was partially extruded when the ion was removed from the medium. The extrusion of Ba2+ from the beta-cell was dependent on external Na+, suggesting that Ba2+ can substitute for Ca2+ in Na+/Ca2+ countertransport. When extracellular Ba2+ was kept between 0.3 and 0.5 mM, large amplitude oscillations (0.1-0.4 min-1) were induced by glucose at concentrations above 7 mM. The oscillations were often transformed into a sustained elevation either by increase of the glucose or Ba2+ concentrations or by the additions of glucagon, forskolin, or carbachol. Although Ba2+ could substitute for Ca2+ in the glucose-induced large amplitude oscillations, there were no Ca(2+)-like pronounced spikes superimposed on an elevated cytoplasmic Ba2+ after elevation of cyclic AMP. Neither could Ba2+ substitute for Ca2+ in being incorporated in response to glucose into a pool mobilizable by carbachol. The studies indicate that cations other than Ca2+ can oscillate in response to glucose, and that such oscillations do not require mobilization from internal pools sensitive to inositol 1,4,5-trisphosphate.

Animals↗

PubMed1994

Caffeine inhibits cytoplasmic Ca2+ oscillations induced by carbachol and guanosine 5'-O-(3-thiotriphosphate) in hyperpolarized pancreatic beta-cells.

The effects of caffeine on cytoplasmic Ca2+ oscillations induced by carbachol and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) were studied in individual mouse pancreatic beta-cells clamped at a hyperpolarized potential. Addition of 10 mM caffeine did not affect the cytoplasmic Ca2+ concentration ([Ca2+]i) in beta-cells exposed to 20 mM glucose and hyperpolarized with diazoxide. Under similar conditions 100 microM carbachol induced a typical response with a marked [Ca2+]i peak followed by a lower sustained elevation. Irrespective of whether 10 mM caffeine was present, there were [Ca2+]i transients with frequencies of 1-5/min superimposed on the sustained phase in 50-60% of the cells. In previously non-exposed cells the introduction of 10 mM caffeine caused temporary lowering of the sustained phase with disappearance of the transients. Subsequent omission of caffeine in the continued presence of carbachol caused a marked [Ca2+]i peak followed by reappearance of the [Ca2+]i transients. However, in cells oscillating in the presence of caffeine its omission caused disappearance of the transients. In this case reintroduction of caffeine restored the transients. In cells kept at -70 mV by a patch pipette containing 100 microM GTP-gamma-S and 3 mM Mg-ATP there were [Ca2+]i transients with frequencies of 0.5-2.5/min. These transients were sufficiently pronounced to activate repetitively a K+ current. Addition of 10 mM caffeine caused disappearance of the [Ca2+]i transients or reduction of their amplitudes and frequencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1994

Glucose induces oscillatory Ca2+ signalling and insulin release in human pancreatic beta cells.

Mechanisms of pulsatile insulin release in man were explored by studying the induction of oscillatory Ca2+ signals in individual beta cells and islets isolated from the human pancreas. Evidence was provided for a glucose-induced closure of ATP-regulated K+ channels, resulting in voltage-dependent entry of Ca2+. The observation of step-wise increases of capacitance in response to depolarizing pulses suggests that an enhanced influx of Ca2+ is an effective means of stimulating the secretory activity of the isolated human beta cell. Activation of muscarinic receptors (1-10 mumol/l carbachol) and of purinergic P2 receptors (0.01-1 mumol/l ATP) resulted in repetitive transients followed by sustained elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i). Periodic mobilisation of intracellular calcium was seen also when injecting 100 mumol/l GTP-gamma-S into beta cells hyperpolarized to -70 mV. Individual beta cells responded to glucose and tolbutamide with increases of [Ca2+]i, manifested either as large amplitude oscillations (frequency 0.1-0.5/min) or as a sustained elevation. Glucose regulation was based on sudden transitions between the basal and the two alternative states of raised [Ca2+]i at threshold concentrations of the sugar characteristic for the individual beta cells. The oscillatory characteristics of coupled cells were determined collectively rather than by particular pacemaker cells. In intact pancreatic islets the glucose induction of well-synchronized [Ca2+]i oscillations had its counterpart in 2-5 min pulses of insulin. Each of these pulses could be resolved into regularly occurring short insulin transients. It is concluded that glucose stimulation of insulin release in man is determined by the number of beta cells entering into a state with Ca(2+)-induced secretory pulses.

Adenosine Triphosphate↗

PubMed1994

Down-regulation of bombesin binding to guinea-pig pancreatic acinar cells during homologous desensitization.

1. [125I]-Tyr4-bombesin exhibited saturable binding to pancreatic acinar cells. 2. Preincubation of cells at 37 degrees C with 0.03 nM-1 microM-bombesin for 10 min followed by acid or neutral washes reduced subsequent binding of [125I]-Tyr4-bombesin in a concentration-dependent manner by up to 90%. 3. In cell suspensions, bombesin raised the cytoplasmic Ca2+ concentration ([Ca2+]i) and the [Ca2+]i response was reduced by pre-exposure to the agonist. 4. In individual superfused cells, bombesin at 1 nM normally caused a large [Ca2+]i transient followed by sustained [Ca2+]i oscillations. The cells exhibited a variable degree of desensitization when subsequently exposed to higher agonist concentrations. 5. Preincubation with bombesin for 10 min caused a concentration-related reduction of subsequent amylase responses to bombesin. 6. Down-regulation of receptor binding is a rapid phenomenon during bombesin exposure explaining, at least partially, tachyphylaxis of [Ca2+]i and amylase responses.

Amylases↗

PubMed1994

Supramaximal inhibition of cholecystokinin-induced pancreatic amylase release involves desensitization to cytoplasmic Ca2+.

BACKGROUND: Cholecystokinin (CCK) is a major stimulant of pancreatic enzyme secretion. The dose-response relationship for CCK-induced secretion is bell-shaped, with a characteristic supramaximal inhibition. The mechanism for this inhibition has now been studied. METHODS: The kinetics of amylase release and the changes of the cytoplasmic Ca2+ concentration ([Ca2+]i) were recorded during stimulation of guinea-pig pancreatic acinar cells with different concentrations of cholecystokinin octapeptide (CCK-8) and the Ca2+ ionophore ionomycin. RESULTS: Individual cells reacted with [Ca2+]i oscillations at 10(-11)-10(-10) M CCK-8 and with an initial peak followed by a sustained suprabasal level at 10(-9)-10(-8) M of the agonist. The latter response was also seen in suspensions of acinar cells at all tested concentrations of CCK-8 and at 10(-6)-10(-5) M of ionomycin. With increases of extracellular Ca2+ from 0.5 to 5.0 mM there was a rise of [Ca2+]i during exposure to 10(-9)-10(-8) M CCK-8 or 10(-5) M ionomycin but a paradoxical decrease at lower concentrations of CCK-8 or ionomycin. A dose-dependent increase of amylase release was seen at CCK-8 concentrations from 10(-11) to 10(-9) M. At 10(-9)-10(-8) M CCK-8 secretion was characterized by an initial peak followed by a sustained phase. Whereas the initial peak of secretion remained unaffected by increasing CCK-8 from 10(-9) to 10(-8) M, the sustained phase was inhibited (supramaximal inhibition). Increasing extracellular Ca2+ from 0.5 to 5.0 mM transiently enhanced secretion in response to 10(-9) M but lacked effect during supramaximal inhibition of secretion by 10(-8) M CCK-8. CONCLUSIONS: Both initial and sustained CCK-8-stimulated amylase release increase with [Ca2+]i. However, supramaximal inhibition of secretion was not due to a decrease of [Ca2+]i but was characterized by desensitization to the stimulatory effect of [Ca2+]i.

Amylases↗

PubMed1993

Dual effects of Na/K pump inhibition on cytoplasmic Ca2+ oscillations in pancreatic beta-cells.

Inhibition of the Na/K pump by ouabain or removal of K+ resulted in gradual increase of intracellular sodium in beta-cell-rich pancreatic islets from ob/ob-mice exposed to 3 mM glucose. In individual beta-cells this action of ouabain was paralleled by closure of ATP-regulated K+ channels and a slow elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i). In most beta-cells an increase of the glucose concentration to 11-20 mM induced large amplitude oscillations of [Ca2+]i with a frequency of 0.2-0.5/min. Ouabain had dual actions on these glucose-induced oscillations in promoting their appearance and at higher concentrations transforming them into a sustained increase of [Ca2+]i. At 100 microM ouabain reduced the frequency of the glucose-induced oscillations but nevertheless raised the time-average [Ca2+]i by increasing the amplitudes and half-widths of the Ca2+ peaks. When high concentrations of ouabain or removal of K+ transformed the oscillations into a sustained increase of [Ca2+]i, the level reached exceeded that obtained in response to rise of glucose alone. By favoring Ca2+ entry and counteracting removal of the cation from the cytoplasm, Na/K pump inhibition perturbs the balance between the processes determining glucose-induced oscillations of [Ca2+]i.

Animals↗

PubMed1992

Cytoplasmic Ca2+ oscillations in pancreatic beta-cells.

In the last 15 years it has been a growing interest in the cyclic variations of circulating insulin [46]. After the suggestion that this phenomenon may be due to oscillations of the beta-cell membrane potential [8,39], it was demonstrated that [Ca2+]i oscillates in the glucose-stimulated beta-cell with a similar frequency to that of pulsatile insulin release. The present review describes four types of [Ca2+]i oscillations in the pancreatic beta-cell. The slow sinusoidal oscillations, referred to as type-a, are those which most closely correspond to pulsatile insulin release. Although not affecting the properties of the type-a oscillations in individual beta-cells, the concentration of glucose is a determinant for their generation and further transformation into a sustained increase. Accordingly, cytoplasmic Ca2+ is regulated by sudden transitions between oscillatory and steady-state levels at threshold concentrations of glucose, which are characteristic for the individual beta-cell. This behaviour explains the observation of a gradual recruitment of previously non-secreting cells with increase of the extracellular glucose concentration [44]. However, it still remains to be elucidated how the sudden transitions between these three states translate into the co-ordinated slow oscillations of [Ca2+]i in the intact islet. Cyclic variations of circulating insulin require a synchronization of the [Ca2+]i cycles also among the islets in the pancreas. It is still an open question by which means the millions of islets communicate mutually to establish a pattern of pulsatile insulin release from the whole pancreas. The discovery that the beta-cell is not only the functional unit for insulin synthesis but also generates the [Ca2+]i oscillations required for pulsatile insulin release has both physiological and clinical implications. The fact that minor damage to the beta-cells prevents the type-a oscillations with maintenance of a glucose response in terms of raised [Ca2+]i reinforces previous arguments [54] that loss of insulin oscillations is an early indicator of type-2 diabetes. Further analyses of the [Ca2+]i oscillations in the beta-cells should include not only the mechanisms for their generation and subsequent propagation within or among the islets but also how modulation of their frequency affects the insulin sensitivity of various target cells. The latter approach may be important in the attempts to maintain normoglycemia under conditions minimizing the vascular effects of insulin supposed to precipitate hypertonia and atherosclerosis [70,71,77].

Animals↗

PubMed1992

Glycine transformation of Ca2+ oscillations into a sustained increase parallels potentiation of insulin release.

Increase of the glucose concentration from 3 to 11 mM resulted in a triphasic release of insulin from perifused ob/ob-mouse beta-cells. A slight inhibition was followed after 2 min by a marked peak and a less pronounced sustained response. At the lower glucose concentration glycine had only marginal effects. However, in the presence of 11 mM glucose, 1-10 mM glycine triggered an immediate and dose-dependent response with an initial peak of insulin release followed by sustained stimulation. In individual beta-cells, rise of the glucose concentration from 3 to 11 mM induced initial lowering of the cytoplasmic Ca2+ concentration ([Ca2+]i) followed by large amplitude oscillations from a level of 50-90 nM to peak values exceeding 300 nM. Already at a concentration of 1 mM, glycine transformed the oscillatory pattern into a sustained level with increase of time-average [Ca2+]i. This elevation became more pronounced in the presence of 10 mM glycine. The effects of glycine on insulin release and [Ca2+]i required extracellular Na+ and were reproduced with the N-methyl analogue sarcosine. It is suggested that glycine potentiation of secretion reflects the elevation of time-average [Ca2+]i both by increased entry and reduced elimination of the cation from the cytoplasm.

Animals↗

PubMed1992

Ga3+ inhibits parathyroid hormone release without interacting with the Ca2+ receptor of the parathyroid cell.

Gallium nitrate is an antihypercalcemic agent with established actions on bone. The effects of Ga(NO3)3 on parathyroid hormone (PTH) release, cytoplasmic Ca2+ concentration ([Ca2+]i) and cAMP production of enzymatically dispersed parathyroid cells from bovine as well as normal and pathological human parathyroid glands have now been studied. Ga3+ at 200 microM inhibited PTH release whereas 600 microM NO3- had no effect. The inhibition was additive to that obtained by elevating extracellular Ca2+. Unlike Ca2+, Ga3+ failed to increase [Ca2+]i or reduce cAMP formation. The results indicate that Ga3+ inhibits PTH release by a mechanism other than activation of the cation receptor of the parathyroid cells. This mechanism may contribute also to inhibition by other cations.

Animals↗

PubMed1992

Effects of gastrin on cytosolic free Ca2+ in individual, acid-secreting rat parietal cells.

The effects of gastrin on cytosolic free Ca2+ ([Ca2+]i) in single, isolated rat gastric parietal cells were investigated using the fluorescent probe Fura-2 and digital image analysis. [Ca2+]i was increased by gastrin (100 nM) in approximately 30% of the parietal cells, which were identified by using either the fluorescent probe acridine orange or a parietal cell-specific monoclonal antibody. In the dominant pattern observed, [Ca2+]i was elevated 50-150% and returned within 1-2 min to a value 30-60% over the basal, which was sustained until withdrawal of the stimulant or addition of the gastrin inhibitor L-365,260 (1 microM). The second, but not the first phase, was abolished in the absence of extracellular Ca2+. The results indicate the existence of functional gastrin receptors in a subpopulation of rat parietal cells.

Acids↗

PubMed1992

Parathyroid-like regulation of parathyroid-hormone-related protein release and cytoplasmic calcium in cytotrophoblast cells of human placenta.

Immunohistochemical staining of human placenta revealed intense reactivity for amino terminal and midregional parathyroid-hormone-related protein (PTHrp) in the cytotrophoblast cells and weaker staining in the syncytiotrophoblasts. The cytotrophoblasts also displayed conspicuous surface staining with the monoclonal antibodies E11 and G11, which recognize a Ca2+ receptor mechanism regulating hormone release of parathyroid cells. Cytotrophoblasts enriched on Percoll gradients or by linking surface-bound E11 to magnetic beads revealed biphasic elevation of cytoplasmic Ca2+ ([Ca2+]i) upon a stepwise rise of external Ca2+ from 0.5 to 3.0 mM, with a half-maximal effect at 1.75 mM. Individual cytotrophoblasts identified by their E11 reactivity disclosed a temporary increase of [Ca2+]i upon elevation of external Mg2+, while Mn2+ triggered both a [Ca2+]i transient and an influx of itself. These effects were efficiently blocked by the G11 antibody. Depolarization with K+ or addition of the voltage-dependent Ca2+ channel blocker verapamil had only marginal effects on [Ca2+]i. Raised extracellular calcium inhibited release of PTHrp from the cells, and this inhibition was blocked by the G11 antibody. The virtually parathyroid-identical Ca2+ regulation of [Ca2+]i may mediate feedback control of PTHrp release from the cytotrophoblasts and thereby participate in the regulation of placental Ca2+ transport.

Calcitriol↗

PubMed1992

BAY K 8644 stimulates glucose-dependent rise of cytoplasmic Ca2+ in hyperpolarized pancreatic beta-cells.

The effect of BAY K 8644 on the cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in pancreatic beta-cells hyperpolarized by the K+ channel-activating agent diazoxide. After 50-60 min preexposure to 0-20 mM glucose in the presence of 400 microM diazoxide [Ca2+]i was close to the level in unstimulated beta-cells. The addition of 5 microM BAY K 8644 then triggered a rise of [Ca2+]i dependent on Ca2+ influx. The magnitude of the BAY K 8644 effect increased with the glucose concentration and was almost 10-fold higher in 20 mM than in the absence of the sugar. It is concluded that glucose can modulate Ca2+ entry through the voltage-dependent channels by a mechanism additional to depolarization. This action may help to explain why previous exposure to the sugar results in an augmented insulin response to a second challenge.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1992

Glucose sensing of individual pancreatic beta-cells involves transitions between steady-state and oscillatory cytoplasmic Ca2+.

Glucose stimulation of individual pancreatic beta-cells is associated with a rise of the cytoplasmic Ca2+ concentration ([Ca2+]i) manifested either as large amplitude oscillations (0.2-0.5/min) or as a sustained increase. Determinants for the transitions between the basal and the two stimulated states have now been studied using dual-wavelength fluorometric measurements on individual ob/ob mouse beta-cells loaded with the Ca2+ indicator Fura-2. The transition from the basal state to large amplitude oscillations was induced by raising the glucose concentration to 7 mM or above. The frequencies and shapes of the [Ca2+]i cycles remained largely unaffected when raising glucose as high as 40 mM. However, in some cells the oscillatory pattern was transformed into a sustained increase of [Ca2+]i at high glucose concentrations. Although the peak values for the oscillations exceeded the steady-state increase, the time average [Ca2+]i was higher during the latter phase. Both types of glucose-induced transitions were facilitated by the presence of 1-100 nM glucagon. Protein kinase C activation by 10 nM of the phorbol ester TPA resulted in a transformation of the glucose-induced oscillations into a sustained increase of [Ca2+]i but the levels reached were considerably lower than obtained with glucose alone. It is concluded that the glucose sensing of the individual beta-cell is based on sudden transitions between steady-state and oscillating cytoplasmic Ca2+. It is these transitions rather than alterations of the oscillatory characteristics which determine the average [Ca2+]i regulating insulin release.

Animals↗